B12 METABOLISM IN HUMANS By NICOLE AURORA LEAL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004
Copyright 2004 by Nicole Aurora Leal
The work in this dissertation is dedicated to my family, especially my parents, for their endless love and support. I would also like to dedicate this dissertation to my fianc, Tearlach Bigger, whose love, patience, and encouragement guided me through this enlightening experience.
ACKNOWLEDGMENTS I would sincerely like to thank my mentor, Dr. Thomas A. Bobik. I find myself continually amazed by his keen intellectual insight and unceasing dedication to research and the furthering of science. I would like to thank the rest of my committee, Dr. Terence R. Flotte, Dr. Nemat O. Keyhani, Dr. Julie A. Maupin-Furlow, and Dr. Keelnatham T. Shanmugam. Their help and guidance along the way were greatly appreciated. I owe a debt of gratitude to Dr. Ruma Banerjee and Horatiu Olteanu, who provided purified human methionine synthase reductase, which was instrumental in the MSR-ATR studies. Special thanks go to Dr. Peter E. Kima for his constant optimism and much-needed expertise in the area of cell biology. Additional thanks go to the entire faculty, staff, and graduate students, especially Stephanie Havemann, at the Microbiology and Cell Science Department. I would also like to extend my appreciation to Dr. Gregory Havemann, Celeste Johnson, Dorothy Park, Edith Sampson, and all the other members of the Bobik lab, for making it a friendly and supportive place to work. The text of Chapter 2 in this dissertation, in part or in full, is a reprint of the material as it appears in the Journal of Biological Chemistry (volume 278, pp. 9227-9234, 2003). The text of Chapter 4 in this dissertation, in part or in full, is a reprint of the material as it appears in Archives of Microbiology (volume 180, pp. 353-361, 2003). iv
TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLE S.............................................................................................................ix LIST OF FIGURES.............................................................................................................x LIST OF ABBREVIATIONS...........................................................................................xii ABSTRACT.......................................................................................................................xv CHAPTER 1 INTRODUCTION........................................................................................................1 History of Cobalamin...................................................................................................1 Structure of Cobalamin.................................................................................................2 Biosynthesis of Cobalamin...........................................................................................3 Cobalamin-Dependent Enzymes..................................................................................4 Cobalamin in Humans..................................................................................................6 Cobalamin-Dependent Enzymes in Humans.........................................................6 Methylmalonyl-CoA mutase..........................................................................7 Methionine synthase.......................................................................................9 Cobalamin Absorption and Transport.................................................................11 Intracellular Cobalamin Metabolism...................................................................12 -ligand transferase......................................................................................12 Cob(III)alamin reductase..............................................................................13 Cob(II)alamin reductase...............................................................................14 Adenosyltransferase.....................................................................................15 Formation of methylcobalamin....................................................................17 Inherited Disorders of Cobalamin Metabolism..........................................................18 Combined Methylmalonic Aciduria and Homocystinuria..................................19 Methylmalonic Aciduria without Homocystinuria..............................................21 Homocystinuria without Methylmalonic Aciduria..............................................23 Salmonella as a Model Organism for the Study of Cobalamin Metabolism..............25 Research Overview.....................................................................................................26 v
2 IDENTIFICATION OF THE HUMAN AND BOVINE ATP:COB(I)ALAMIN ADENOSYLTRANSFERASE CDNAS BASED ON COMPLEMENTATION OF A BACTERIAL MUTANT........................................................................................36 Introduction.................................................................................................................36 Materials and Methods...............................................................................................38 Chemicals and Reagents......................................................................................38 Bacterial Strains and Growth Media...................................................................39 General Protein and Molecular Methods and ATR Assays.................................39 P22 Transductions...............................................................................................39 Screening the Bovine and Human Liver cDNA Libraries...................................40 Cloning the Bovine Adenosyltransferase Coding Sequence for High-Level Expression...............................................................................40 Cloning the Human Adenosyltransferase Coding Sequence for High-Level Expression...............................................................................41 Cloning the Human Adenosyltransferase Coding Sequence for Complementation Studies..........................................................................42 Growth of Adenosyltransferase Expression Strains and Preparation of Cell Extracts................................................................................................43 Growth Curves.....................................................................................................43 Western Blots......................................................................................................44 DNA Sequencing and Analysis...........................................................................45 Results.........................................................................................................................45 Screening of the Bovine and Human Liver cDNA Libraries for Clones that Express Adenosyltransferase Activity.............................................................45 Identification of a Human Gene Related to the Bovine Adenosyltransferase cDNA...............................................................................................................47 Putative Subcellular Localization of Adenosyltransferase Enzymes..................48 High-Level Expression of the Bovine and Human Adenosyltransferases in E. coli...........................................................................................................48 Assay of the Bovine and Human Adenosyltransferase Fusion Proteins for ATR Activity..............................................................................................49 Complementation of an S. enterica Mutant Deficient in ATR Activity by a Human Adenosyltransferase cDNA Clone......................................................50 Level of Human Adenosyltransferase Enzyme in Normal and cblB Mutant Human Skin Fibroblasts...................................................................................51 Conserved Amino Acids and Distribution of Adenosyltransferase Enzymes.....52 Discussion...................................................................................................................53 3 PURIFICATION AND INITIAL CHARACTERIZATION OF THE HUMAN ATP:COB(I)ALAMIN ADENOSYLTRANSFERASE AND ITS INTERACTION WITH METHIONINE SYNTHASE REDUCTASE.................................................62 Introduction.................................................................................................................62 Materials and Methods...............................................................................................64 Chemicals and Reagents......................................................................................64 Bacterial Strains and Growth Media...................................................................65 vi
General Molecular and Protein Methods.............................................................65 ATP:cob(I)alamin Adenosyltransferase Assays..................................................65 Construction of ATR Expression Strains............................................................66 Purification of the Human ATR Variants............................................................66 MSR-ATR Assay.................................................................................................68 Measurement of Cob(I)alamin with Iodoacetate.................................................68 Separation and Quantification of Cobalamins by HPLC....................................68 Effect of Ionic Strength on the MSR-ATR System.............................................69 DNA Sequencing.................................................................................................69 Results.........................................................................................................................70 Purification of the Human ATR Variants............................................................70 Linearity of the ATR reaction.............................................................................71 ATR Reaction Requirements...............................................................................71 Alternative Nucleotide Donors............................................................................71 Km and Vmax Values for ATR 239K and 239M...................................................71 MSR Reduces Cob(II)alamin to Cob(I)alamin for AdoCbl Synthesis................72 Cob(I)alamin is Sequestered by the MSR-ATR System.....................................73 MSR Produces little Cob(I)alamin in the Absence of the ATR..........................75 Stoichiometry of the MSR-ATR system.............................................................75 Ionic Strength Dependence of Cobalamin Reduction and Adenosylation by the MSR-ATR System................................................................................75 Discussion...................................................................................................................76 4 PDUP IS A COENZYME-A-ACYLATING PROPIONALDEHYDE DEHYDROGENASE ASSOCIATED WITH THE POLYHEDRAL BODIES INVOLVED IN B12-DEPENDENT 1,2-PROPANEDIOL DEGRADATION BY SALMONELLA ENTERICA SEROVAR TYPHIMURIUM LT2...............................92 Introduction.................................................................................................................92 Materials and Methods...............................................................................................95 Bacterial Strains, Media, and Growth Conditions...............................................95 General Molecular Methods................................................................................95 General Protein Methods.....................................................................................95 P22 Transduction.................................................................................................96 Cloning pduP into pLAC22.................................................................................96 Construction of His8-PduP..................................................................................97 Purification of His8-PduP under Denaturing and Nondenaturing Conditions.....97 Propionaldehyde Dehydrogenase Assays............................................................98 Identification of Propionyl-CoA a Product of the Propionaldehyde Dehydrogenase Reaction.................................................................................99 Construction of a Nonpolar pduP Deletion.......................................................100 Antibody Preparation.........................................................................................101 Western Blots....................................................................................................101 Electron Microscopy.........................................................................................102 DNA Sequencing and Analysis.........................................................................102 Chemicals and Reagents....................................................................................102 Results.......................................................................................................................103 vii
Effect of a Precise pduP Deletion on the Growth of S. enterica on Minimal 1,2-Propanediol Medium...............................................................................103 Propionaldehyde Dehydrogenase Activity in Wild-type S. enterica and a pduP Mutant.......................................................................................105 High-level Production of the PduP Protein.......................................................105 Purification of Recombinant His8-PduP Protein...............................................106 Propionyl-CoA is a Product of the PduP Reaction............................................107 Preparation and Specificity of the Anti-PduP Antiserum..................................108 Localization of PduP Immunoelectron Microscopy..........................................109 Propionaldehyde Dehydrogenase Activity is associated with Purified pdu Bodies...............................................................................110 Discussion.................................................................................................................110 5 CONCLUSIONS......................................................................................................119 Identification of the Bovine and Human Adenosyltransferase.................................119 Biochemical Characterization of the Human Adenosyltransferase..........................120 Methionine Synthase Reductase is a Cob(II)alamin Reductase for the Human Adenosyltransferase.....................................................................121 Future Experimentation............................................................................................123 LIST OF REFERENCES.................................................................................................125 BIOGRAPHICAL SKETCH...........................................................................................139 viii
LIST OF TABLES Table page 1-1. Adenosylcobalaminand methylcobalamin-dependent reactions.............................30 2-1. Bacterial strains.........................................................................................................56 2-2. Specific activities of bovine and human ATP:cob(I)alamin ATRs...........................59 3-1. ATP:cob(I)alamin adenosyltransferase activity during ATR 239K and 239M purification...............................................................................................................82 3-2. Use of alternative nucleotide donors by the human ATR.........................................84 3-3. The MSR-ATR system sequesters cob(I)alamin.......................................................85 4-1. Bacterial strains.......................................................................................................113 4-2. Propionaldehyde dehydrogenase activity in extracts from wild-type S. enterica and strain BE191 (pduP)......................................................................................114 4-3. Propionaldehyde dehydrogenase activity during polyhedral body purification......115 ix
LIST OF FIGURES Figure page 1-1. Structure of cobalamins.............................................................................................29 1-2. Different classes of cobalamin-dependent enzymes..................................................31 1-3. Pathways of propionyl-CoA catabolism and cobalamin metabolism........................32 1-4. Pathways involving methionine synthase and the reductive activation of methionine synthase by methionine synthase reductase..........................................33 1-5. Cobalamin metabolism in mammalian cells, and disorders associated with deficiencies in this pathway.....................................................................................34 1-6. Proposed pathway of AdoCbl-dependent 1,2-propanediol degradation in S. enterica.............................................................................................................35 2-1. Multiple sequence alignment of adenosyltransferase enzymes.................................57 2-2. SDS-PAGE analysis of cell extracts from bovine and human ATR expression strains.....................................................................................................58 2-3. Complementation of an ATR-deficient bacterial mutant for AdoCbl-dependent growth on 1,2-propanediol by a plasmid that expresses the human ATR................60 2-4. Western blot analysis of ATR expression by normal and cblB mutant cell lines.....61 3-1. Propionyl-CoA metabolism, methionine synthesis, and intr acellular cobalamin metabolism...............................................................................................................81 3-2. Purification of the human ATR.................................................................................83 3-3. Determination of kinetic constants for ATR 239K and ATR 239M.........................86 3-4. Absorbance spectra of an MSR-ATR assay..............................................................87 3-5. Two schemes depicting the conversion of cob(II)alamin to AdoCbl by the MSR-ATR system....................................................................................................88 3-6. MSR produces little cob(I)alamin in the absence of the ATR enzyme.....................89 x
3-7. Stoichiometry of the MSR-ATR system...................................................................90 3-8. Effect of ionic strength on the MSR-ATR system....................................................91 4-1. SDS-PAGE analysis of His8-PduP..........................................................................116 4-2. Western blot using the anti-PduP antiserum............................................................117 4-3. Immunogold localization of the PduP enzyme........................................................118 xi
LIST OF ABBREVIATIONS A adenine ADP adenosine diphosphate AdoCbl adenosylcobalamin AIM aldehyde indicator medium Amp ampicillin ATP adenosine triphosphate ATR adenosyltransferase BSA bovine serum albumin C cytosine C degree centigrade Cam chloramphenicol cbl cobalamin cDNA complementary deoxyribonucleic acid CH3Cbl methylcobalamin CH3THF methyl tetrahydrofolate CNCbl cyanocobalamin, Vitamin B12 CMCbl carboxymethylcobalamin ddH20 distilled deionized water DMB dimethylbenzimidazole DNA deoxyribonucleic acid xii
DTT dithiothreitol E. coli Escherichia coli FAD flavin adenine dinucleotide FldA flavodoxin FMN flavin mononucleotide Fre flavin oxidoreductase Fpr flavodoxin reductase G guanine GSCbl glutathionylcobalamin HOCbl hydroxycobalamin IF intrinsic factor IFCR intrinsic factor cobalamin receptor IPTG isopropyl--D-thiogalactopyranoside Kan kanamycin kDa kiloDaltons Km Michaelis Constant for enzyme activity LB Luria Bertani medium MCM methylmalonyl-CoA mutase MTS mitochondrial targeting sequence min minutes mg milligram mM millimolar MS methionine synthase xiii
MSR methionine synthase reductase NADPH nicotinamide adenine dinucleotide phosphate â€“ reduced form NCE no carbon E medium OD optical density PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction SAM S-adenosylmethionine S. enterica Salmonella enterica SDS sodium dodecyl sulfate T thymine TCII transcobalamin II TCII-R transcobalamin II receptor TCA tricarboxylic acid THF tetrahydrofolate Tris tris hydroxymethyl aminomethane tRNA transfer ribonucleic acid U uracil V volt Vmax maximal rate of enzyme activity XCbl inactive cobalamin derivatives X-Gal 5-bromo-4-chloro-3-indolyl--D-galactopyranoside xiv
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy B12 METABOLISM IN HUMANS By Nicole Aurora Leal August 2004 Chair: Thomas A. Bobik Major Department: Microbiology and Cell Science In humans, the B12 coenzymes, adenosylcobalamin and methylcobalamin, are required cofactors for propionate metabolism and methionine biosynthesis, respectively. Humans are incapable of de novo synthesis of the B12 coenzymes and require complex precursors such as vitamin B12 in their diet. The metabolism of vitamin B12 to adenosylcobalamin requires two successive reductions and an adenosylation reaction; however, many aspects of the genetics and biochemistry of this process have not been elucidated. This dissertation focuses on the isolation and characterization of the human genes and enzymes involved in the formation of adenosylcobalamin from vitamin B12. Deficiencies in this process result in methylmalonic aciduria, a rare disorder that is often fatal in newborns. Because of the sophisticated genetic methods that are available, Salmonella enterica was used as a model system. By complementation of an S. enterica mutant, a bovine adenosyltransferase cDNAs was isolated and subsequent sequence similarity xv
searches identified a homologous human cDNA. The bovine and human cDNAs were independently cloned, expressed in Escherichia coli, and the encoded proteins were shown to have adenosyltransferase activities similar to previously studied bacterial adenosyltransferases. Additional studies, using Western blots, showed that adenosyltransferase expression was altered in cell lines derived from patients with cblB methylmalonic aciduria when compared to cell lines from normal individuals. Two common human adenosyltransferase polymorphic variants were expressed in E. coli, purified, and biochemically characterized. Both had high specificity for ATP and comparable Km and Vmax values. An adenosyltransferase-linked cob(II)alamin reductase assay was developed and used to show that the human methionine synthase reductase functions as a cob(II)alamin reductase for adenosylcobalamin formation. A linked assay was also performed in the presence of a cob(I)alamin trapping agent, and results from these experiments suggested that human adenosyltransferase and methionine synthase reductase interact. Importantly, results from this work provide information that will allow improvements in diagnosis and treatment of methylmalonic aciduria, a devastating childhood disorder. xvi
CHAPTER 1 INTRODUCTION History of Cobalamin In 1925, Whipple and colleagues discovered that a diet of liver aided in the formation of blood cells in iron-deficient dogs. The next year, Minot and Murphy (Minot and Murphy 1926) were the first to document that patients with severe pernicious anemia (a disease that affects the formation of red blood cells) were successfully treated with a special diet consisting mainly of liver. Eight years later (in 1934) Whipple, Minot, and Murphy were awarded the Nobel Prize in Medicine and Physiology for their work on liver therapy against anemias. Following these studies, Castle (Kass 1978) concluded that the treatment of pernicious anemia by this specialized diet was dependent on the absorption of an extrinsic anti-pernicious anemia factor by an intrinsic protein in gastric juice. It is now known that Castleâ€™s extrinsic anti-pernicious anemia factor is vitamin B12 and the intrinsic factor is a specific cobalamin transporter (synthesized by the stomach) that was shown to enhance the curing effect by vitamin B12 (Kass 1978). In 1948, two research groups led by Folkers and Smith independently isolated the red crystalline anti-pernicious anemia factor from bovine liver, and proposed the name vitamin B12 (Rickes et al. 1948, Smith and Parker 1948). The isolation of vitamin B12 inspired collaborative efforts between two laboratories (led by Alexander Todd and Dorothy Hodgkin) to elucidate its chemical structure. After 6 years, the crystal structure was resolved and finalized in the 1
2 laboratory of Dorothy Hodgkin (Hodgkin et al. 1955), a feat for which she was awarded the 1964 Nobel Prize in Chemistry. Today we know that two active cobalamin derivatives are required for enzyme catalysis. One is the adenosyl derivative (adenosylcobalamin) which was identified in 1958, when Barker and coworkers reported the isolation of a light-sensitive cobalamin compound that was a required cofactor for glutamate mutase (Barker et al. 1958). Shortly after the discovery of this compound, its crystal structure was resolved by Lenhert and Hodgkin (Lenhert and Hodgkin 1961). The other biologically active cobalamin derivative was discovered later as methylcobalamin, a required cofactor for methionine synthase (Guest et al. 1962a, Guest et al. 1962b). The structure of methylcobalamin was resolved decades later by Jenny Gluskers laboratory (Rossi et al. 1985). Structure of Cobalamin Cobalamin is the largest and most complex cofactor known to man. It is a water-soluble molecule composed of three general structural units: a central ring, an upper ligand, and a lower ligand (Figure 1-1). At the core of cobalamin is a ring structure designated as corrin, which is synthesized from uroporphyrinogen III, the last common intermediate of heme, siroheme, chlorophyll, and cobalamin synthesis. The corrin ring consists of four reduced pyrrole rings (A, B, C, and D). Unlike heme and chlorophyll (in which the pyrrole rings are linked by four methylene bridges), cobalamin has a direct bond between rings A and D. In cobalamin, the four nitrogen atoms of the pyrrole rings form coordinate bonds with a central cobalt atom; whereas the central atom of the tetrapyrrole ring is iron and magnesium, in heme and chlorophyll, respectively. Below the plane of the corrin ring is the -ligand of the cobalt atom, dimethylbenzimidazole
3 (DMB). The DMB moiety is covalently attached to the corrin ring by a ribose phosphate bonded to the aminopropanol side chain on ring D and is also coordinated to the central cobalt atom. The exact function of the lower ligand is not completely understood, but has been suggested that it may have a role in coenzyme binding and catalysis (Banerjee and Ragsdale 2003). Above the plane of the corrin ring is the -ligand of the cobalt atom. The -ligand varies in the different forms of cobalamins and includes cyano, hydroxy, glutathionyl, methyl, and adenosyl moieties. Cyanocobalamin (vitamin B12, CNCbl) is an artifact obtained during isolation of cobalamins from natural sources and is the most common pharmaceutical form (Rickes et al. 1948). CNCbl together with hydroxycobalamin (HOCbl), and glutathionylcobalamin (GSCbl) are precursors for synthesis of the biologically active coenzymes, methylcobalamin (CH3Cbl) and adenosylcobalamin (AdoCbl). Biosynthesis of Cobalamin Cobalamin or closely related corrinoid compounds are required in all kingdoms of life with perhaps the exception of plants and fungi. De novo synthesis of B12 is restricted to some Bacteria and Archaea (Roth et al. 1996), requiring all other life forms that use B12 to obtain complex precursors from their diet. Characterized as the most complex nonpolymeric natural substance known, cobalamin is synthesized de novo by a multi-step enzymatic process consisting of uroporphyrinogen III synthesis, side chain modification, cobalt insertion, aminopropanol addition, DMB biosynthesis, and addition of methyl or adenosyl moieties (Battersby 1994). The microbial biosynthesis of cobalamin can proceed along two different pathways: one aerobic and the other anaerobic (Scott 2003). These pathways differ both in their requirement for molecular oxygen and the timing of
4 cobalt insertion. Pseudomonas denitrificans and Bacillus megaterium use the aerobic pathway of cobalamin synthesis whereby molecular oxygen is used to assist with ring contraction and cobalt is inserted late in the synthesis. In contrast, cobalamin synthesis by the anaerobic pathway (used by Salmonella enterica and Propionibacterium freundenreichii) is prevented by the presence of molecular oxygen, and cobalt is inserted early during synthesis. Unlike prokaryotes, humans are unable to synthesize cobalamin de novo and are dependent on a dietary source of a complex precursor such as vitamin B12 from which AdoCbl and CH3Cbl are synthesized (Baker and Mathan 1981). Cobalamin-Dependent Enzymes AdoCbl and CH3Cbl share structural and chemical similarities; however, they catalyze very different biochemical reactions. The weak covalent carbon-cobalt bond of cobalamin, which is highly reactive and crucial for enzyme catalysis, is a shared trait of both cofactors (Frey and Reed 2000). In AdoCbl-dependent reactions, carbon-based free radicals are generated by the homolytic cleavage of the carbon-cobalt bond (Halpern 1985); while in methyltransferase reactions, the carbon-cobalt bond of CH3Cbl is heterolytically cleaved, releasing a methyl group and cob(I)alamin (Banerjee 1997). AdoCbl-dependent enzymes can be classified into four types: eliminases (including dehydratases and deaminases), amino mutases, carbon skeleton mutases, and ribonucleotide reductases. The first three types are isomerases that catalyze 1,2-rearrangement reactions (Table 1-1). Their main use is in the fermentation of small molecules and they use the same basic catalytic mechanism (Banerjee 1999). The reaction is triggered by substrate binding, causing homolytic cleavage of the carbon-cobalt bond, thereby generating an adenosyl radical and cob(II)alamin. The adenosyl radical abstracts a hydrogen atom from the substrate, forming a substrate radical
5 and deoxyadenosine. After hydrogen abstraction, rearrangement of the substrate radical results in product radical formation. The product radical then abstracts a hydrogen atom from deoxyadenosine, forming both product and an adenosyl radical. Finally, the adenosyl radical recombines with cob(II)alamin, reforming the coenzyme (which can be used in additional catalytic cycles). The last type of AdoCbl-dependent enzymes is ribonucleotide reductases, which generate deoxyribonucleotides for DNA synthesis. There are three classes of ribonucleotide reductases. Class I ribonucleotide reductases are strict aerobic enzymes and are divided into two subclasses (Class Ia and Ib). Class Ia ribonucleotide reductases are found in eukaryotes, plants, viruses, and some prokaryotes. These enzymes contain three conserved cysteines, a tyrosyl radical, and a nonheme diiron center which are essential for catalysis (Aberg et al. 1989, Nordlund and Eklund 1993). Class Ib ribonucleotide reductases are found in prokaryotes and vary from class Ia in their lack of allosteric regulation and the use of different electron carriers for ribonucleotide reduction (Jordan et al. 1994). The Class II ribonucleotide reductases require AdoCbl, are found in bacteria and archaea and are active both aerobically and anaerobically. Their reaction mechanism is similar to that of the isomerase reaction, differing where the adenosyl radical does not interact directly with the substrate, but with a cysteinal radical in the active site of the enzyme (Booker et al. 1994). Class III ribonucleotide reductases are oxygen sensitive and are found in some prokaryotes and methanogens (Reichard 1993). Although this class has not been well characterized it is thought that a glycyl radical, an iron sulfur center and S-adenosylmethionine (SAM) are required for catalysis (Harder et al. 1992).
6 CH3Cbl-dependent enzymes are involved in methyl transfer reactions (Table 1-1). Only the reaction mechanisms of methionine synthase and the acetyl-CoA synthase have been studied extensively (Ljungdahl and Wood 1982, Jarrett et al. 1998); however, all of the methyltransferases are thought to follow the same general mechanism. The reaction involves three steps, and begins with the methylation of enzyme-bound cob(I)alamin by a methyl-group donor, forming enzyme-bound CH3Cbl. The carbon-cobalt bond of CH3Cbl is then heterolytically cleaved, resulting in enzyme-bound cob(I)alamin and a methyl cation, which is transferred to the substrate forming the methylated product. Methyltransferases are essential for methionine synthesis and are also involved in acetate and methane synthesis (Schneider and Stroinski 1987). Figure 1-2 shows the different types of B12-dependent enzymes and the reactions they catalyze. Cobalamin in Humans Humans are incapable of de novo synthesis of cobalamin, and must take up complex cobalamin precursors from the diet. Cobalamin is a water-soluble vitamin that cannot freely diffuse across the intestinal wall because of its large size and requires specific carriers and transporters for uptake and delivery to cells. Once in the cells, cobalamin is then metabolized to CH3Cbl in the cytosol and AdoCbl in the mitochondria, by a series of enzyme-mediated reactions. Cobalamin-Dependent Enzymes in Humans In humans, AdoCbl and CH3Cbl are required for two enzymes that are essential in metabolism (Kolhouse and Allen 1977). One of these enzymes is methylmalonyl-CoA mutase (MCM), an AdoCbl-dependent enzyme used to metabolize propionyl-CoA resulting from the breakdown of odd-chain fatty acids, cholesterol, and some amino acids. The other is methionine synthase (MS), a CH3Cbl-dependent enzyme used for
7 homocysteine recycling and methionine synthesis. Deficiencies in MCM or MS can lead to methylmalonic aciduria or homocystinuria (rare but often lethal childhood disorders). Methylmalonyl-CoA mutase MCM is the only AdoCbl-dependent enzyme that is present in both bacterial and mammalian systems. In humans, it resides in the mitochondrial matrix, and is used for the metabolism of propionyl-CoA to succinyl-CoA. In this metabolic pathway, propionyl-CoA is carboxylated to (2S)-methylmalonyl-CoA, isomerized to (2R)-methylmalonyl-CoA, and lastly rearranged to succinyl-CoA by MCM (Figure 1-3). In contrast, in some bacteria including Propionibacterium shermanii, MCM is used in the opposite metabolic flow to produce propionate from succinate (Allen et al. 1964). The gene encoding MCM has been sequenced and cloned from many organisms including human, P. shermanii, mouse, Streptomyces cinnamonensis, Porphyromonas gingivalis, and Sinorhizobium meliloti (Banerjee and Chowdhury 1999). In humans, the location of the MCM gene was mapped to chromosome 6p12-21.2, and the cDNA was shown to have a mitochondrial targeting sequence (MTS) (Ledley et al. 1988). In native conformation, the human and bacterial MCMs are 150 kiloDaltons (kDa), and are dimeric proteins. The crystal structure of P. shermanii MCM has been determined, and is an heterodimer that binds one mole of AdoCbl (Marsh et al. 1989). The human MCM is a homodimer of two subunits that binds two moles of AdoCbl and has 60% sequence identity to the chain of the bacterial MCM (Mancia et al. 1996). UV, visible, and electron paramagnetic resonance spectroscopic studies have shown that AdoCbl binding by MCM occurs via â€œBase-offâ€ (class I) interaction (Padmakumar and Banerjee 1995). â€œBase-offâ€ binding occurs by the displacement of the
8 DMB ligand by a histidine residue on the -subunit of MCM. DMB is still attached to the corrin ring through a nucleotide loop; however, in the class I interaction, the coordinate bond of DMB to cobalt is displaced and substituted by His610 of the -subunit. The exact role of the His610 residue is not fully understood, but current studies suggest that its major role is in DMB displacement and organization of the active site for AdoCbl binding and enzyme catalysis (Vlasie et al. 2002). The isomerization of methylmalonyl-CoA to succinyl-CoA by MCM requires homolytic cleavage of the carbon-cobalt bond of enzyme-bound AdoCbl (Padmakumar and Banerjee 1997). Homolysis of the organometallic bond occurs once methylmalonyl-CoA binds MCM. This results in the formation of an adenosyl radical and cob(II)alamin. The carbon-cobalt bond of free AdoCbl is weak and in solution has a rate of homolysis of 4x10-10 sec-1. With the addition of MCM, the rate of homolysis is increased to higher than 600 sec-1, suggesting that MCM promotes the homolysis of AdoCbl for this rearrangement reaction (Padmakumar and Banerjee 1997). The adenosyl radical generated by homolysis abstracts hydrogen from the methyl-group of methylmalonyl-CoA, forming a primary-substrate radical and 5â€™-deoxyadenosine. The primary-substrate radical undergoes an intramolecular 1,2-rearrangement resulting in the formation of a secondary product radical that abstracts hydrogen from deoxyadenosine. Lastly, there is recombination of the adenosyl radical and cob(II)alamin, regenerating AdoCbl and releasing succinyl-CoA that can flow into the tricarboxylic acid (TCA) cycle. In humans, defects in the enzyme MCM cause a block in propionyl-CoA metabolism, resulting in a buildup of methylmalonyl-CoA that is hydrolyzed to
9 methylmalonic acid. Methylmalonic acid is excreted into the urine, resulting in methylmalonic aciduria, an inherited disorder that is often fatal in newborns. Methionine synthase MS catalyzes the conversion of CH3THF and homocysteine to THF and methionine. This enzyme is found in both bacteria and humans. In E. coli, two MS enzymes occur and they differ in their requirement for CH3Cbl (Drummond and Matthews 1993). In humans, only CH3Cbl-dependent MS is present. The E. coli and human genes encoding CH3Cbl-dependent MS have been cloned and encode large monomeric proteins of 136 and 141 kDa, respectively (Banerjee et al. 1989, Leclerc et al. 1996). The human gene was mapped to chromosome 1 at position q43 (Leclerc et al. 1996). The bacterial and the human enzymes share 55% identity in amino acid sequence, and have established similarities in the mechanism of catalysis (Hall et al. 2000). However, only the E. coli CH3Cbl-dependent MS has been studied extensively. Methionine synthase from E. coli is a modular enzyme consisting of four domains, all of which are crucial for enzyme catalysis (Goulding et al. 1997). The N-terminal domain (residues 2-353) has been cloned, expressed, and shown to be involved in homocysteine binding, and methyl group transfer from CH3Cbl to homocysteine (Goulding et al. 1997). It contains three cysteine residues used to coordinate a zinc ion essential for homocysteine binding, which are conserved in other MS enzymes including Homo sapiens, Caenorhabditis elegans, and Synechocystis species (Goulding and Matthews 1997). The second domain (residues 354-649) was shown to catalyze methyl transfer from CH3THF to cob(I)alamin (Goulding et al. 1997). The third domain (residues 650-896) was lacking enzymatic activity; however, it was shown to be essential for CH3Cbl binding (Banerjee et al. 1989). This fragment was crystallized and shown to
10 bind CH3Cbl in the class I â€œBase-offâ€ mode (Drennan et al. 1994). The last domain (residues 897-1227), termed the activation domain, was shown to bind SAM required for the reductive activation of MS (Dixon et al. 1996, Hall et al. 2000). The mechanism of MS catalysis and reactivation is shown in Figure 1-4. The methyltransferase reaction requires heterolytic cleavage of the carbon-cobalt bond of CH3Cbl resulting in cob(I)alamin and a methyl group. After heterolysis, MS transfers its methyl group to homocysteine, forming methionine and MS-bound cob(I)alamin. During catalysis, additional methyl groups are provided by methyltetrahydrofolate (CH3THF), which regenerates CH3Cbl and releases tetrahydrofolate (THF) (Banerjee and Matthews 1990). Occasionally, the MS-bound cob(I)alamin is oxidized to cob(II)alamin during catalytic turnover, and reductive activation is required to restore the methylation cycle (Fujii et al. 1977, Banerjee et al. 1990, Drummond and Matthews 1993). This involves reduction of cob(II)alamin and methylation by SAM to form CH3Cbl. The reduction of cob(II)alamin for MS activation differs in E. coli and humans. Reduced flavodoxin (FldA) provides the needed electron in E. coli (Fujii and Huennekens 1974). However, humans lack flavodoxin and instead use the dual flavoprotein methionine synthase reductase (MSR) (Olteanu and Banerjee 2001). In humans, a block in the methylation cycle caused by defects in MS results in homocystinuria, a severe and sometimes fatal childhood disease (Fenton and Rosenberg 2000). In addition, partial defects in MS can lead to elevated homocysteine, a major cardiovascular disease risk factor (Refsum et al. 1998). A deficiency in MS also results in the trapping of cellular folate as CH3THF, making it unavailable for other
11 folate-dependent reactions including purine and pyrimidine biosynthesis (Wilson et al. 1999). Cobalamin Absorption and Transport As mentioned above, humans are incapable of de novo cobalamin synthesis, and require complex precursors in their diet. Suitable precursors (such as HOCbl or CNCbl) can be obtained by the consumption of beef, liver, poultry, fish, eggs, dairy products, and vitamin supplements (Stabler 1999). The cobalt of these cobalamin molecules is in the +3 oxidation state (cob(III)alamin), the form that is recognized for cobalamin absorption and transport. Once ingested, cobalamin molecules that are bound to food proteins are released by the combined action of proteases and acid in the stomach (Del Corral and Carmel 1990). Haptocorrin (a cobalamin carrier protein) binds released cobalamin and transports it from the stomach to the small intestines. Haptocorrin has a high specificity for cobalamin and when bound to cobalamin, protects it from damage by acids in the stomach, until it reaches the small intestines where cobalamin is liberated from haptocorrin via digestion by pancreatic enzymes. After being released from haptocorrin, cobalamin is bound by intrinsic factor (IF), a glycoprotein produced by the parietal cells (gastric glands lining the stomach). The IF-cobalamin complex is resistant to further digestion in the small intestines because of the carbohydrates on the IF. The IF-cobalamin complex recognizes the IF-cobalamin receptor (IFCR) on the epithelial cells of the distal third small intestines (ileum), and is transported into these cells via receptor-mediated endocytosis. In the epithelial cell, IF is degraded by the acidic environment of the lysosome, and cobalamin is released. Transcobalamin II (TCII), a serum-transport protein in the epithelial cells, binds released cobalamin, and transports it out of the cell and into the bloodstream until it is taken up by other cells. The
12 TCII-cobalamin complex binds the TCII receptor on the target cell, and gains entry via endocytosis where it can be used for intracellular cobalamin metabolism (Rosenblatt and Fenton 1999, Seetharam 1999, Watkins and Rosenblatt 2001). Intracellular Cobalamin Metabolism Once the TCII-cobalamin complex is internalized by the target cell, the transport protein is degraded in the lysosomal vesicle, and cobalamin is released from this complex. A cobalamin transporter then shuttles free cobalamin from the lysosome to the cytosol of the cell. Intracellular cobalamin metabolism (Figure 1-5) is thought to be similar in prokaryotes and eukaryotes (Qureshi et al. 1994, Watkins and Rosenblatt 2001). In the cytosol, cob(III)alamin, in the form of HOCbl or CNCbl, is converted to GSCbl and then reduced to cob(II)alamin. These steps are proposed to be catalyzed by a -ligand transferase and a cob(III)alamin reductase, respectively. After cobalamin reduction in the cytosol, cob(II)alamin is localized to the mitochondrial matrix and is reduced to cob(I)alamin by a mitochondrial cob(II)alamin reductase. The final step in AdoCbl formation is the adenosylation of cob(I)alamin to form AdoCbl by an ATP:cob(I)alamin adenosyltransferase. The pathways of AdoCbl and CH3Cbl synthesis are shared, and thought to branch apart after the reduction of cob(III)alamin. For CH3Cbl metabolism, cytosolic cob(II)alamin associates with MS, forming MS-bound-cob(II)alamin which is reduced to cob(I)alamin by MSR and methylated by SAM to form MS-CH3Cbl. -ligand transferase The first step in cobalamin metabolism is removal of the -ligand. Cobalamin -ligand transferase activity has been detected in crude cell extracts of Clostridium
13 tetanomorphum, P. shermanii, Euglena gracilis, human leukocytes, human skin fibroblasts, rat liver, and rabbit spleen (Weissbach et al. 1961, Brady et al. 1962, Watanabe et al. 1987, Pezacka et al. 1990, Pezacka 1993). The enzymes involved and their encoding genes have not been identified. However, it was determined that for the enzymatic conversion of CNCbl to AdoCbl, cell extracts required supplementation with adenosine triphosphate (ATP), reduced flavin, and reduced glutathione (Weissbach et al. 1962). Studies in mammalian cells suggest that GSCbl is a product of the -ligand transferase reaction (Pezacka et al. 1990). In bacteria, this GSCbl intermediate has not been found. Cob(III)alamin reductase The second enzymatic step in cobalamin metabolism is cob(III)alamin reduction. Reductase activity has been detected in crude cell extracts of C. tetanomorphum, P. shermanii, E. gracilis, human, and rat (Weissbach et al. 1961, Brady et al. 1962, Watanabe et al. 1987, Pezacka et al. 1990, Pezacka 1993). Extracts of C. tetanomorphum required NADH and either flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) to mediate reductase activity (Walker et al. 1969). The flavin oxidoreductase (Fre) of S. enterica was purified and shown to mediate the nonenzymatic reduction of cobalamin in vitro (Fonseca and Escalante-Semerena 2000). Fre reduces flavin nucleotides, which in turn chemically reduces cob(III)alamin. In E. gracilis, cob(III)alamin reductase activity was purified from the mitochondrial fraction, and was ultimately identified as a flavoprotein with an absolute requirement for NADPH but not FAD or FMN (Watanabe et al. 1987). Further analysis of this protein revealed that it contained one molecule of either FAD or FMN that remained bound during the
14 purification, explaining why cob(III)alamin reductase activity was retained without the addition of these flavins. Cell extracts from human fibroblasts and rat liver have cob(III)alamin reductase activity associated with both microsomes and the mitochondrial membrane, that was derived from oxidation-reduction reactions by cytochromes and flavoproteins (Watanabe et al. 1989, Watanabe et al. 1996). There has been no genetic evidence that the flavoproteins studied in these systems are the physiological cob(III)alamin reductases. The studies by Watanabe and colleagues also indicate that both prokaryotic and eukaryotic systems produce redundant enzymes with the ability to reduce cobalamin; however, this reduction could be a primarily nonenzymatic process. Cob(II)alamin reductase Cob(I)alamin is one of the strongest nucleophiles known to exist in aqueous solution, and is a powerful reductant (Eâ€™ = -0.61 V) (Banerjee et al. 1990). Because of the nucleophilic nature of cob(I)alamin, cob(II)alamin reduction is an energetically unfavorable reaction. Thus, it is no surprise that cob(II)alamin reductase activity can only be studied by coupling the reaction to an alkylation event such as adenosylation or methylation. Cob(II)alamin reductase activity has been detected in cell-free extracts of C. tetanomorphum and P. shermanii; however, these enzymes have not been identified, and the encoding genes are unknown (Brady et al. 1962, Weissbach et al. 1962, Walker et al. 1969). In humans, cob(II)alamin reductase activity has been detected in mitochondrial fractions, but the enzyme was never identified, and the gene is unknown (Fenton and Rosenberg 1978b). Purification of the cob(II)alamin reductase from cell extracts of C. tetanomorphum did not produce a single enzyme, but produced a complex containing the adenosylating
15 enzyme. This suggests that cob(II)alamin reductase and the adenosyltransferase may exist as a structural complex under certain conditions (Walker et al. 1969). Advantages of this complex would decrease the probability of unfavorable cob(I)alamin side reactions and instead allow for adenosylation because of the close proximity of cob(I)alamin to the adenosyltransferase. The E. coli FldA protein has been shown to function as a cob(II)alamin reductase for the formation of AdoCbl (Fonseca and Escalante-Semerena 2001). In vitro, flavodoxin reductase (Fpr), flavodoxin (FldA), and purified CobA (an adenosyltransferase) catalyze AdoCbl synthesis from cob(III)alamin (Fonseca and Escalante-Semerena 2001). In this coupled assay, it is proposed that Fpr uses NADPH to reduce FldA, which then reduces cob(III)alamin to cob(I)alamin while bound to CobA (Fonseca and Escalante-Semerena 2001). To date, there are no reports showing an interaction between FldA and CobA. Although this reducing system is functional in vitro, there is no genetic evidence that FldA is the physiological cob(II)alamin reductase (Fonseca and Escalante-Semerena 2001). Adenosyltransferase Adenosylation is the terminal step in AdoCbl metabolism. ATP:cob(I)alamin adenosyltransferase (ATR) activity was detected in crude cell extracts of human fibroblast cells, but the enzyme involved and the encoding gene were never identified (Fenton and Rosenberg 1981). ATRs from P shermanii, C. tetanomorphum, P. denitrificans, Thermoplasma acidophilum, and S. enterica have been purified and partially characterized (Brady et al. 1962, Vitols et al. 1966, Debussche et al. 1991, Suh and Escalante-Semerena 1995, Johnson et al. 2001, Saridakis et al. 2004). The genes encoding these enzymes have been identified from P. denitrificans, T. acidophilum, and
16 S. enterica (Crouzet et al. 1991, Suh and Escalante-Semerena 1993, Johnson et al. 2001, Saridakis et al. 2004). To date, three families of adenosyltransferases have been identified: EutT-type, PduO-type, and CobA-type. These families are classified based on amino acid sequence similarity and examples of each are found in S. enterica. EutT is thought to be involved in the conversion of CNCbl to AdoCbl for ethanolamine utilization (Kofoid et al. 1999). PduO adenosylates cob(I)alamin for AdoCbl synthesis which is required for B12-dependent 1,2-propanediol metabolism (Johnson et al. 2001). CobA can substitute for PduO in cob(I)alamin adenosylation for 1,2-propanediol metabolism, but its primary role is the adenosylation of cobalamin intermediates for de novo B12 biosynthesis (Suh and Escalante-Semerena 1995, Johnson et al. 2001). The CobA-type and PduO-type ATRs have been extensively studied and well characterized biochemically. CobA ATR from S. enterica has been overexpressed and purified (Suh and Escalante-Semerena 1995). This enzyme was shown to have a broad specificity for reduced corrinoid substrates, which could be due to its role in de novo cobalamin synthesis (Suh and Escalante-Semerena 1995). The X-ray crystal structure of CobA in its free state, complexed with ATP and HOCbl has been resolved (Bauer et al. 2001). It is a homodimer, which is consistent with the subunit composition of CobO ATR from P. denitrificans (Debussche et al. 1991, Bauer et al. 2001). The N-terminal P-loop (GNGKGKT) of CobA coordinates the -, -, and -phosphates of ATP, however the crystal structure shows that this interaction occurs in the opposite orientation when compared to other P-loop dependent nucleotide hydrolases (Bauer et al. 2001). This unique mode of ATP binding positions the 2â€™-OH group to allow for nucleophilic attack
17 at the 5â€™-C of ATP by cob(I)alamin resulting in adenosylation (Fonseca et al. 2002). Additionally, triphosphate was identified as a reaction byproduct of adenosylation and was also found to be a strong inhibitor of the reaction (Fonseca et al. 2002). The archaeal T. acidophilum PduO-type ATR has been purified, crystallized, and partially characterized (Saridakis et al. 2004). T. acidophilum ATR has 27% amino acid identity to PduO. This ATR is specific for ATP and deoxy-ATP. Additionally, the crystal structure revealed a trimeric configuration. Each of the subunits is composed of five alpha helical domains that aid in subunit assembly due to hydrophobic, ionic, and polar interactions depending on the domain. In contrast to CobA, there is no recognizable P-loop motif suggesting a different binding method for ATP. Formation of methylcobalamin The synthesis CH3Cbl is coupled to cob(II)alamin reduction (Huennekens et al. 1982). Once cob(II)alamin is reduced, methylation is dependent on SAM as the methyl donor for the formation of CH3Cbl. The E. coli cob(II)alamin reductase requires FldA for the reductive activation of MS (Fujii and Huennekens 1974). Humans lack FldA, requiring an alternate reductase for CH3Cbl formation. Because of the role of FldA in bacteria, it was proposed that the human reductase would contain binding sites for FMN and FAD (Leclerc et al. 1998). Using homology based PCR, a cDNA with consensus sequences to predicted binding sites for FMN, FAD, and NADPH was cloned and proposed to encode methionine synthase reductase (MSR), the cob(II)alamin reductase required for MS activation (Leclerc et al. 1998). The MSR gene (MTRR) was mapped to chromosome 5 at position p15.2 (Leclerc et al. 1999). Analysis of the gene revealed that the N-terminal sequence is consistent with cytosolic targeting, but alternative splicing at the 5â€™ end of MTRR mRNA generates a
18 mitochondrial targeting sequence (Leclerc et al. 1999). There have been no studies documenting the role of MSR in the mitochondria. Banerjee and Olteanu (2001) showed that MSR is a monomeric protein of 77 kDa, that when purified contains stoichiometric amounts of FAD and FMN. In an NADPH-dependent reaction, purified MSR reduced cob(II)alamin to cob(I)alamin for activation of MS in vitro (Olteanu and Banerjee 2001). In these same studies, it was found that the reduction of cobalamin bound to MS by MSR is dependent on electrostatic interactions. Inherited Disorders of Cobalamin Metabolism In humans, inherited disorders in the metabolic pathways of both AdoCbl and CH3Cbl synthesis result in methylmalonic aciduria and homocystinuria. Methylmalonic aciduria is caused by a block in the pathway of propionyl-CoA metabolism which causes an accumulation methylmalonyl-CoA that is hydrolyzed to methylmalonic acid, and excreted into the blood and urine (Rosenblatt and Cooper 1990). Clinically, methylmalonic aciduria presents itself by lethargy, recurrent vomiting, dehydration, respiratory distress, feeding difficulties, failure to thrive, hypotonia, and death (Ciani et al. 2000). Homocystinuria is the result of a block in the methylation cycle for methionine synthesis, and results in an accumulation of homocysteine, which is excreted into the blood and urine (Rosenblatt and Cooper 1990). Homocystinuria presents itself by megaloblastic anemia, delayed development, neurological disorders, cardiovascular disease, and death (Kapadia 1995). All of the disorders involved in cobalamin metabolism are inherited as autosomal recessive traits (Rosenblatt and Fenton 1999). There have been no reported heterozygotes with the presentation of these diseases. Inborn errors of cobalamin metabolism have been classified into eight distinct complementation groups, cblA to cblH (Figure 1-5). This classification is based on
19 biochemical studies and complementation analysis of human cultured fibroblast cells (Fenton and Rosenberg 1978a). Complementation analysis examines the uptake and conversion of labeled precursors (propionate, CH3THF) by fused and unfused fibroblast cell lines derived from two patients with cobalamin disorders. In these studies, fused cells that stimulated the incorporation of labeled precursors complemented each other and were assigned to different complementation groups. If the fused cells were unable to stimulate incorporation of the precursors they were assigned to the same complementation group (Gravel et al. 1975). An assay that has helped to determine if defects are due to decreased synthesis of one or both cobalamin coenzymes involves incubating the cultured fibroblasts with labeled HOCbl and measuring the cofactors formed against control cell lines. Combined Methylmalonic Aciduria and Homocystinuria Defects in cellular cobalamin metabolism resulting in both methylmalonic aciduria and homocystinuria are defined by the complementation groups cblF, cblC, and cblD. Cultured fibroblasts from patients in these groups were analyzed and shown to have a deficiency in the conversion of CNCbl or HOCbl to AdoCbl or CH3Cbl cofactors (Qureshi et al. 1994). The absence of these cofactors renders both MCM and MS inactive. There are over 100 patients with combined methylmalonic aciduria and homocystinuria. The cblC complementation group has more than 100 patients, the cblD group has only two patients who are siblings, and the cblF group has six patients (Watkins and Rosenblatt 2001). Individuals with the cblF disorder have impaired MCM and MS activities. The total intracellular pools of AdoCbl and CH3Cbl are reduced when compared to control cells. Cultured cells incubated with [57Co]-labeled CNCbl showed an accumulation of
20 unmetabolized, non-protein bound CNCbl contained within the lysosomes (Qureshi et al. 1994). There was no synthesis of either AdoCbl or CH3Cbl in these cells. This disorder is defined by a deficiency in the release of cobalamin from the lysosomes after endocytosis making cobalamin unavailable to intracellular enzymes (Kapadia 1995). The gene encoding cobalamin lysosomal transport has not been identified. Other clinical presentations of this disorder include hypotonia, stomatitis, facial abnormalities, developmental retardation, and failure to thrive (Fenton and Rosenberg 2000). Individuals diagnosed with the cblC and cblD disorders have very similar biochemical characteristics. Both of these groups have decreased total cobalamin content in the liver, kidney, and fibroblasts (Kapadia 1995). Cultured cells from these patients incubated with [57Co]-labeled CNCbl or HOCbl have reduced conversion to labeled AdoCbl or CH3Cbl when compared to controls (Mahoney et al. 1971). Because of the reduced levels of these cofactors, MCM and MS activities are deficient in cultured cells even though the enzymes are normal (Mellman et al. 1978). In light of these results, it is proposed that cblC and cblD complementation groups result from a defect in cytosolic cob(III)alamin reductase activity (Watkins and Rosenblatt 2001). Based on complementation studies, the cblC and cblD groups are classified as mutations occurring at unique chromosomal locations; however, the specific role of these two groups has not been determined and their corresponding genes remain unidentified. The clinical presentation of the cblC disorder is more severe than cblD. In addition to methylmalonic aciduria and homocystinuria, the cblC group usually presents itself in the neonatal stage (early-onset) resulting in megaloblastic anemia, congenital malformations, lethargy, failure to thrive, poor feeding, and death (Rosenblatt et al.
21 1997). Both cases of the cblD disorders did not develop problems until later on in life (late-onset) resulting in megaloblastic marrow, neuromuscular problems, methylmalonic aciduria, homocystinuria, and mental retardation (Rosenblatt and Cooper 1990). Since the cblD group is biochemically similar to cblC but presents itself as less severe, it is suggested that cblD is a leaky form of cblC, or that it may be affecting an unidentified step before or after the reduction of cobalamin (Qureshi et al. 1994). The cblC, cblD, and cblF groups respond to pharmacological doses of HOCbl which reduces methylmalonic aciduria and homocystinuria but does not eliminate them (Fenton and Rosenberg 2000). Methylmalonic Aciduria without Homocystinuria Defects in the cellular metabolism of cobalamin resulting in methylmalonic aciduria without homocystinuria are defined by cblA, cblB, and cblH complementation groups. Complementation analysis of fibroblast cells from these patients has shown that the cblA, cblB, and cblH defects occur at three distinct chromosomal locations (Qureshi et al. 1994, Watkins et al. 2000). Patients with these disorders have a deficiency in intracellular AdoCbl levels and have normal CH3Cbl levels (Watkins and Rosenblatt 2001). There are at least 45 patients diagnosed with cblA, 36 individuals with cblB, and only one reported individual with cblH disorder (Watkins and Rosenblatt 2001). The cblA and cblH complementation groups are deficient in mitochondrial AdoCbl synthesis. Cultured cells from these patients were unable to convert [57Co]-labeled HOCbl to labeled AdoCbl but had no abnormality in CH3Cbl synthesis (Fenton and Rosenberg 2000). Cell extracts from cblA patients required a reducing system for the synthesis of AdoCbl; however, intact mitochondria from these patients were unable to synthesize AdoCbl even in the presence of a reducing system. On the other hand, control intact mitochondria can synthesize AdoCbl from HOCbl without the addition of reducing
22 agents (Fenton and Rosenberg 1978b), suggesting that the cblA disorder could be due to a deficiency in cobalamin reduction or abnormalities of mitochondrial cobalamin binding and transport (Mahoney et al. 1975). The gene encoding the cblA disorder was identified (MMAA) and shown to localize to chromosome 4q31.1-2 (Dobson et al. 2002). It appears that this gene product has mitochondrial functionality due to the presence of an N-terminal MTS. Dobson and colleagues suggested that the MMAA protein does not encode a mitochondrial cobalamin reductase because it lacks NADPH, and flavin binding motifs that would be essential for reductase activity (Watanabe et al. 1987), (Dobson et al. 2002). The MMAA protein has 61% similarity to ArgK in E. coli, an accessory protein common to lysine (L), arginine (A), ornithine (O), and LAO transport systems (Dobson et al. 2002). It is thought that ArgK could have some function in B12 transport in E. coli and has been proposed that MMAA could encode a mitochondrial cobalamin transporter (Dobson et al. 2002). Recently, MeaB, a homolog of the human MMAA, from Methylobacterium extorquens AM1 was identified (Korotkova et al. 2002). Homology searches (using MeaB and MCM) identified a fusion protein from Burkholderia fungorum with an N-terminal MeaB domain and a C-terminal MCM domain (Korotkova and Lidstrom 2004). The MCM and MeaB homologue fusion protein in B. fungorum suggests that these two proteins would interact in systems where the genes are expressed separately (Korotkova and Lidstrom 2004). It has been shown that the M. extorquens MeaB exists as a complex with MCM (Korotkova and Lidstrom 2004). It is now thought that the cblA group (MMAA) encodes a protein that would interact with MCM and protect it from inactivation (Korotkova and Lidstrom 2004).
23 The cblB complementation group is characterized by a deficiency in the conversion of cobalamin to AdoCbl. Cell extracts derived from patients with cblB disorder were unable to synthesize AdoCbl even when provided with a reducing system, labeled HOCbl, and ATP (Fenton and Rosenberg 1981). Patients with this disorder are defective in ATP:cob(I)alamin ATR activity. At the start of this study, the gene encoding the cblB defect was unknown and the protein had not been isolated. The clinical presentation of cblA, cblB, and cblH disorders are very similar and include methylmalonic aciduria, lethargy, failure to thrive, vomiting, dehydration, respiratory distress, and anemia (Fenton and Rosenberg 2000). These disorders have been treated by a diet restricted in amino acid precursors of methylmalonate and cobalamin supplementation (Kapadia 1995). Homocystinuria without Methylmalonic Aciduria Inborn errors of cellular cobalamin metabolism resulting in homocystinuria without methylmalonic aciduria are defined by cblE and cblG complementation groups. There has been only one patient in the cblE group that had combined homocystinuria and mild methylmalonic aciduria (Wilson et al. 1999). Cultured fibroblasts from these patients show a deficiency in CH3Cbl synthesis and decreased methionine synthase activity (Watkins and Rosenblatt 1989). Complementation analysis has shown that cblE and cblG defects occur at distinct chromosomal locations (Watkins and Rosenblatt 1988). Fibroblasts from patients with cblE and cblG disorders showed normal incorporation of labeled propionate into macromolecules; however, the incorporation of labeled CH3THF was reduced when compared to controls suggesting an abnormality in the methionine synthase pathway (Watkins and Rosenblatt 1989). To date there are 12 patients
24 diagnosed with the cblE disorder and 28 individuals with the cblG disorder (Fenton and Rosenberg 2000). Members of the cblE complementation group are deficient in methionine synthase activity due to a block in CH3Cbl formation. Fibroblast extracts from patients with this disorder had normal methionine synthase activity when high concentrations of reducing agent were added to the assay, but activity was reduced or absent when the reducing agent was used at lower concentrations (Watkins and Rosenblatt 1988). From these studies it was shown that the cblE group is defective in cobalamin reduction. The gene corresponding to this group was identified (MTRR) and localized to chromosome 5p15.2-p15.3 (Leclerc et al. 1998). The MTRR protein or MSR is required for the reductive activation of methionine synthase and was reviewed above (Wilson et al. 1999). The cblG complementation group is also deficient in methionine synthase activity but has normal levels of CH3Cbl. Fibroblast extracts from these patients were assayed and showed no MS activity under optimal conditions (Watkins and Rosenblatt 1989). This group is defined by defects in MS. Human MS is a modular enzyme with domains containing binding sites for homocysteine, CH3THF, SAM, and cob(II)alamin (Banerjee 1997). The cDNAs from cblG mutants have been analyzed and shown to contain missense and nonsense mutations (Leclerc et al. 1996). It appears that a majority of deficiencies in the cblG group are due to the lack of MS protein (caused by a premature stop codon due to nonsense mutations) and not in cobalamin binding. The clinical presentations of the cblE and cblG groups include homocystinuria without methylmalonic aciduria, developmental delay, megaloblastic anemia, and neurological disorders
25 (Watkins et al. 2002). Some patients respond to treatment with HOCbl (Watkins and Rosenblatt 2001). Salmonella as a Model Organism for the Study of Cobalamin Metabolism Salmonella enterica is an important model organism for studies of B12-dependent processes (Schneider and Stroinski 1987, Roth et al. 1996). More is known about cobalamin metabolism and physiology in S. enterica than in any other single organism. Cobalamin transport, cobalamin biosynthesis, intramolecular rearrangements, and methyl transfer reactions have been investigated in S. enterica (Roth et al. 1996). In addition, S. enterica has a well-defined genetic system that includes methods for transformation, transduction, gene expression, directed, chemical, and transposon mutagenesis and a known genome sequence. One of the best-studied AdoCbl dependent enzymes in S. enterica is diol dehydratase. This enzyme is crucial for the metabolism of 1,2-propanediol. The genes required for growth of S. enterica on 1,2-propanediol are organized at the propanediol utilization (pdu) locus. Determination of the DNA sequence indicated that this locus has 23 genes (Bobik et al. 1997, Bobik et al. 1999): six pdu genes are thought to encode enzymes needed for the 1,2-propanediol degradative pathway (pduCDEPQW) (Bobik et al. 1997); two are involved in transport and regulation (pduF and pocR) (Bobik et al. 1992, Chen et al. 1994); two are probably involved in diol dehydratase reactivation (pduGH) (Bobik et al. 1999); one is needed for the conversion of cobalamin to AdoCbl (pduO) (Johnson et al. 2001); five are of unknown function (pduLMSVX); and seven share similarities to genes involved in the formation of carboxysomes (pduABJKNTU), polyhedral bodies found in certain cyanobacteria and chemoautotrophs (Shively and English 1991, Shively et al. 1998). The large number of genes required for the
26 degradation of propanediol suggests that this compound serves an important purpose in the lifestyle of Salmonella (Roth et al. 1996). Salmonella uses propanediol as a carbon and energy source in a B12-dependent manner. The proposed pathway of propanediol degradation was determined based on biochemical studies (Figure 1-6). The pathway begins with the conversion of 1,2-propanediol to propionaldehyde, a reaction catalyzed by the AdoCbl-dependent diol dehydratase (Toraya et al. 1979, Obradors et al. 1988). Propionaldehyde is then disproportionated to propanol and propionic acid by a reaction series thought to involve propanol dehydrogenase, phosphotransacylase, and propionate kinase (Toraya et al. 1979, Obradors et al. 1988). Aerobic growth of S. enterica on 1,2-propanediol requires addition of CNCbl (or other complex cobalamin precursors) to growth media. Research Overview Coenzyme B12-dependent processes are essential for human health. Methylmalonyl-CoA mutase and methionine synthase, the only two reported B12-dependent enzymes in humans, are crucial for propionate metabolism and methionine synthesis. Inborn errors of cobalamin metabolism can block these pathways resulting in methylmalonic aciduria and homocystinuria, serious diseases that are often fatal in newborns. Cobalamin metabolism has been studied in both bacterial and eukaryotic systems, and has been found to be quite similar. Progress in identifying human genes involved in B12 metabolism has been slow due to difficulties in purifying the corresponding enzymes and the lack of facile genetic techniques. To circumvent these problems, S. enterica was used as a model system for studies of B12 metabolism in humans.
27 Chapter 2 of this dissertation reports the identification of the human ATP:cob(I)alamin adenosyltransferase involved in AdoCbl metabolism. An S. enterica mutant deficient in ATR activity allowed for the isolation of a bovine ATR cDNA. Subsequent sequence similarity searching was used to identify a homologous human cDNA. Both the bovine and human cDNAs were independently cloned and overexpressed in E. coli. Enzyme assays showed that both the bovine and human enzymes had ATR activity in vitro. Subsequent studies showed that the human cDNA clone complemented an ATR-deficient S. enterica strain for AdoCbl-dependent growth on 1,2-propanediol in vivo. In addition, Western blots were used to show that ATR expression is altered in cell lines derived from cblB methylmalonic aciduria patients compared with cell lines from normal individuals. Chapter 3 of this dissertation reports the biochemical properties of the human ATR and its interaction with methionine synthase reductase. Two common polymorphic variants of the ATR, which are found in normal individuals, were expressed in E. coli and purified to apparent homogeneity. Purified ATR variants were used for kinetic studies and the catalytic properties of both ATR variants including the Vmax and the Km for ATP and cob(I)alamin were determined. Investigations also showed that the purified methionine synthase reductase in combination with purified ATR can convert cob(II)alamin to AdoCbl in vitro. In this system, MSR reduced cob(II)alamin to cob(I)alamin which was adenosylated to AdoCbl by the ATR enzyme and results indicated that MSR and ATR interact in such a way that the highly reactive intermediate (cob(I)alamin) was sequestered. The finding that MSR can reduce cob(II)alamin to cob(I)alamin for AdoCbl synthesis (in conjunction with the prior finding that MSR
28 reduced cob(II)alamin for the activation of methionine synthase) indicates a dual physiological role for the MSR enzyme. Chapter 4 of this dissertation is not directly related to B12 metabolism in humans, but was the research performed during my first two years in Dr. Bobikâ€™s Laboratory. This work focused on the identification of PduP, a coenzyme-A-acylating propionaldehyde dehydrogenase associated with polyhedral bodies involved in AdoCbl-dependent 1,2-propanediol degradation by S. enterica. A PCR based method was used to construct a precise nonpolar chromosomal deletion of the gene pduP. The resulting pduP deletion strain grew poorly on 1,2-propanediol minimal media and expressed 105-fold less propionaldehyde dehydrogenase activity than did wild-type S. enterica grown under similar conditions. An E. coli strain was constructed for high-level production of His8-PduP, which was purified by nickel-affinity chromatography and was shown to have propionaldehyde dehydrogenase activity. Reverse-phase High Performance Liquid Chromatography (HPLC) and mass spectrometry were used to verify the product of the PduP reaction. Immunogold electron microscopy was used to show that PduP is associated with a polyhedral organelle involved in AdoCbl-dependent 1,2-propanediol degradation.
29 Co3+NCCCCNCCCCNCCHCCNCCCCCCH3H3CH3CCOHNH2NCOH2NCOH3CH2NCOCH3CCH3CH3CNH2OCNH2OH3CCNH2OHCCH3OPOOONNOOHH3CH3CCH2OHCHRCorrin RingDimethylbenzimidazoleAminopropanolADCBR= upper -ligand NNNNNH2OOHOHCH2CNRRRCH3R = A c t i v e R = I n a c t i v e AdenosylAdoCblMethylCH3CblCyanoCNCbl,vitamin B12HydroxyHOCblROHGlutathionylGSCblRGS Co3+NCCCCNCCCCNCCHCCNCCCCCCH3H3CH3CCOHNH2NCOH2NCOH3CH2NCOCH3CCH3CH3CNH2OCNH2OH3CCNH2OHCCH3OPOOONNOOHH3CH3CCH2OHCHRCorrin RingDimethylbenzimidazoleAminopropanolADCBR= upper -ligand NNNNNH2OOHOHCH2CNRRRCH3R = A c t i v e R = I n a c t i v e AdenosylAdoCblMethylCH3CblCyanoCNCbl,vitamin B12HydroxyHOCblROHGlutathionylGSCblRGS Figure 1-1. Structure of cobalamins. The major parts of cobalamin are labeled: upper -ligand, corrin ring, aminopropanol, and dimethylbenzimidazole. The four pyrrole rings of the corrin are labeled with capital letters. The upper ligand can be any of the groups listed in the legend. Active and inactive forms of cobalamin are dependent on the upper ligand.
30 Table 1-1. Adenosylcobalaminand methylcobalamin-dependent reactions AdoCbl-dependent Isomerases MeCbl-dependent Methyltransferases Diol dehydratase: 1,2-propanediol propionaldehyde + H20 Methionine synthase: homocysteine CH3THF methionine + THF Glycerol dehydratase: glycerol -hydroxypropionaldehyde + H20 Ethanolamine ammonia lyase: ethanolamine acetaldehyde + H20 L--Lysine aminomutase: L--lysine L-erythro-3,5-diaminohexanoic acid Acetyl -CoA synthesis via Corrinoid/Fe protein: (a) CH3THF + corrinoid/FeS protein THF + CH3-corrinoid Fe/S protein (b) CH3-corrinoid Fe/S protein + carbon monoxide corrinoid Fe/S protein + acetyl-CoA D--Lysine aminomutase: D--lysine 2,5-diaminohexanoic acid D-Ornithine aminomutase: D-ornithine 2,4-diaminovaleric acid Methyltetrahydromethanopterin:coenzyme M methyltransferase: CH3-H4MPT + coenzyme M H4MPT + CH3-coenzyme M Methylmalonyl-CoA mutase: methylmalonyl-CoA succinyl-CoA Glutamate mutase: glutamate -methylaspartate Methanol:2-mercaptoethanesulfonic acid methyltransferase: CH3OH + coenzyme M CH3-coenzyme M + H20 Isobutyryl-CoA mutase: isobutyryl-CoA butyryl-CoA Methyleneglutarate mutase: 2-methylene glutarate 3-methylitaconate Ribonucleotide reductase: NTP dNTP
31 OHOH1,2-propanediolOpropionaldehydediol dehydrataseAdoCblH2O+H2NCOHNH2L--lysine 5,6 aminomutaseAminomutasesDehydratasesethanolamine ammonia lyaseCarbon skeleton mutasesRibonucleotide reductasesMethyltransferases(P)PPOBASEOOHOHHHHHNDP, NTPmethylmalonyl-CoAsuccinyl-CoAethanolamineacetaldehydeNH3H2NOHCHH3COOHOCHCCH3COSCoAHOCCH2CH2COSCoA+methionine synthaseCHCH2CH3THFTHFCH3Cblcob(I)alaminmethylmalonyl CoA-mutaseHOCCH2NH2OhomocysteinemethionineOOSHCHCH2HOCCH2NH2OSCH3ClassesDeaminasesAdoCblAdoCblAdoCblAdoCblribonucleotide reductase-lysineL-erythro 3,5 diaminohexanoic acidH3CCOHNH2ONH2ReactionsdNDP, dNTP(P)PPOBASEOHOHHHHHEliminases Figure 1-2. Different classes of cobalamin-dependent enzymes. The enzymes involved and the reactions they catalyze are highlighted.
32 CH2COH3CSCoApropionyl-CoACHCOCH3SCoAHOOC(2S)-methylmalonyl-CoApropionyl-CoA carboxylaseBiotin, HCO32-,ATP, Mg2+(2R)-methylmalonyl-CoAmethylmalonyl-CoA epimeraseCH2COSCoAHOOCCH2succinyl-CoACHCOOHCH3HOOCmethylmalonyl-CoA mutasemethylmalonic acidValineIsoleucineMethionineThreonineOdd chain fatty acidsThymineCholesteroladenosylcobalaminHCCOSCoAHOOCCH3CENTRAL METABOLISMHOC b lGSCblcob(II)alamincob(I)alamin-ligand transferasecob(III)alaminreductasecob(II)alaminreductaseadenosyltransferase Figure 1-3. Pathways of propionyl-CoA catabolism and cobalamin metabolism. Propionyl-CoA carboxylase catalyzes the formation of (2S)-methylmalonyl-CoA. Methylmalonyl-CoA epimerase catalyzes the conversion of (2S)-methylmalonyl-CoA to the (2R) isomer. AdoCbl-dependent methylmalonyl-CoA mutase catalyzes the conversion of (2R)-methylmalonyl-CoA to succinyl-CoA. In humans, a deficiency in MCM or in any of the metabolic steps needed for AdoCbl synthesis leads to methylmalonic aciduria.
33 MS-cob(I)alaminMS-CH3CblhomocysteinemethionineTHFCH3THFMS-cob(II)alaminSAMS A HMS-CH3CblMethionine SynthaseMethionine SynthaseReductaseA)B) Figure 1-4. Pathways involving methionine synthase and the reductive activation of methionine synthase by methionine synthase reductase. A) Proposed pathway of methionine synthesis. In this pathway, CH3Cbl-dependent MS transfers a methyl from CH3THF to homocysteine forming methionine. B) Reductive activation of MS-bound cob(II)alamin. MSR reduces cobalamin, and SAM provides a methyl for the reactivation of MS which reenters the methylation cycle.
34 TCIIHOCbl LysosomeTCIIHOCbl TCIIHOCbl HOCblCob(II)alaminCob(II)alaminCob(I)alaminAdenosylcobalaminMethylmalonyl-CoA Mutase mutMethylmalonyl-CoASuccinyl-CoA MitochondrioncblFcblC,cblDcblAcblBGSCbl-ligand transferase AdenosyltransferaseCob(II)alamin Reductase CytoplasmCob(III)alamin Reductase MS-Cob(II)alaminHomocysteineMethionine cblGMethionine SynthaseMS-Cob(I)alaminCH3THFMS-CH3Cbl SAMMS ReductasecblE cblH TCIIHOCbl LysosomeTCIIHOCbl TCIIHOCbl HOCblCob(II)alaminCob(II)alaminCob(I)alaminAdenosylcobalaminMethylmalonyl-CoA Mutase mutMethylmalonyl-CoASuccinyl-CoA MitochondrioncblFcblC,cblDcblAcblBGSCbl-ligand transferase AdenosyltransferaseCob(II)alamin Reductase CytoplasmCob(III)alamin Reductase MS-Cob(II)alaminHomocysteineMethionine cblGMethionine SynthaseMS-Cob(I)alaminCH3THFMS-CH3Cbl SAMMS ReductasecblE cblH Figure 1-5. Cobalamin metabolism in mammalian cells, and disorders associated with deficiencies in this pathway. The pathways by which inactive cobalamin precursors are taken up and metabolized to active cofactors are shown. The cytoplasmic and mitochondrial compartments are labeled. The specific steps affected by inborn errors of cobalamin metabolism (mut, cblA cblH) are labeled.
35 OHOH1,2-propanediolOpropionaldehydediol dehydratase(PduCDE)OHpropanolOSCoApropanoldehydrogenase(PduQ)propionyl-CoAOPO32-propionyl-phosphateOOpropionateATPADPCoA-dependent propionaldehyde dehydrogenase(PduP)phosphotransacylasepropionate kinase(PduW)NADH + H+NAD+adenosylcobalaminNADH + H+NAD+adenosyltransferase(PduO)XCbl Figure 1-6. Proposed pathway of AdoCbl-dependent 1,2-propanediol degradation in S. enterica. AdoCbl-dependent diol dehydratase catalyzes the conversion of 1,2-propanediol to propionaldehyde. Propionaldehyde is then disproportionated into propanol and propionate presumably by aldehyde dehydrogenase (PduP), phosphotransacylase, propionate kinase (PduW) and propanol dehydrogenase (PduQ). When grown aerobically, S. enterica is unable to synthesize cobalamin de novo and requires cobalamin precursors (XCbl) for the metabolism to AdoCbl.
CHAPTER 2 IDENTIFICATION OF THE HUMAN AND BOVINE ATP:COB(I)ALAMIN ADENOSYLTRANSFERASE CDNAS BASED ON COMPLEMENTATION OF A BACTERIAL MUTANT Introduction Enzymes dependent on the vitamin B12 coenzymes, AdoCbl and CH3Cbl have a broad but uneven distribution among the three domains of life (Dolphin 1982, Schneider and Stroinski 1987, Banerjee 1999). In higher animals, there are two known cobalamin-dependent enzymes. CH3Cbl dependent MS is needed for the methylation of homocysteine to methionine (Cauthen et al. 1966, Drummond and Matthews 1993), and AdoCbl-dependent MCM plays an essential role in the conversion of propionyl-CoA to the TCA cycle intermediate, succinyl-CoA (Banerjee and Chowdhury 1999, Matthews 1999). This latter process occurs in three steps: propionyl-CoA is carboxylated to (2S)-methylmalonyl-CoA, isomerized to (2R)-methylmalonyl-CoA, and finally rearranged to succinyl-CoA in a reaction catalyzed by AdoCbl-dependent MCM (Figure 1-3). In higher animals, propionyl-CoA is produced from the breakdown of the amino acids valine, isoleucine, methionine, and threonine, as well as thymine, cholesterol and odd-chain fatty acids; hence, MCM is essential for the complete catabolism of each of these compounds (Banerjee and Chowdhury 1999). In humans, inherited defects that impair the activity of AdoCbl-dependent MCM lead to methylmalonic aciduria, a rare but severe disease that is often fatal in the first year of life (Rosenblatt and Fenton 1999, Fenton et al. 2000, Olteanu and Banerjee 2001). Such inherited deficiencies can result from mutations in the MCM structural gene (mut) 36
37 or from mutations that impair the acquisition of the required cofactor, AdoCbl (Rosenblatt and Fenton 1999, Watkins and Rosenblatt 2001). Higher animals are incapable of de novo synthesis and, hence, must obtain AdoCbl by synthesis from complex precursors taken up from their diet (Banerjee 1999). Suitable precursors include vitamin B12 (CNCbl) and other cobalamins with various -ligands (XCbls) (Banerjee 1999). The pathway by which XCbls are converted to AdoCbl has been studied in several organisms and is thought to be similar in both prokaryotes and eukaryotes (Figure 1-3): XCbl is converted to glutathionyl-cobalamin (GSCbl), reduced to cob(II)alamin, further reduced to cob(I)alamin and finally adenosylated to AdoCbl (Friedmann 1975, Huennekens et al. 1982, Pezacka et al. 1990, Pezacka 1993, Watanabe et al. 1996). In humans, four complementation groups (cblABCD), associated with methylmalonic aciduria, are thought to correspond to genes involved in the conversion of XCbls to AdoCbl (Figure 1-5). Enzymatic assays of fibroblast extracts have indicated that the cblC and cblD complementation groups encode cytoplasmic enzyme(s) needed for the conversion of XCbls to cob(II)alamin (Mellman et al. 1979). Similar studies have indicated that the cblA and cblB complementation groups correspond to a mitochondrial cob(II)alamin reductase and an ATP:cob(I)alamin ATR enzymes, respectively (Mahoney et al. 1975, Fenton and Rosenberg 1981). To date, the human genes that correspond to the cblABCD complementation groups have not been identified. Progress in this area has been slow due to the difficulties in purifying the relevant proteins (Rosenblatt and Cooper 1990). We recently identified the PduO ATR of Salmonella, and showed that this enzyme has partial functional redundancy with the CobA enzyme (Johnson et al. 2001). In these
38 studies, it was also shown that a Salmonella pduO cobA double mutant lacked AdoCbl-dependent diol dehydratase activity due to the ATR deficiency. Furthermore, it was found that a plasmid-encoded source of ATR enzyme restored diol dehydratase activity to a cobA pduO double mutant (Johnson et al. 2001). Because this enzymatic activity can be readily detected on aldehyde indicator medium (AIM), we expected that the ATR-deficient Salmonella strain could be used to screen expression libraries for cDNAs that encode ATR enzymes. Such an approach would circumvent the difficulties associated with ATR purification. Here, we report the isolation of an ATR cDNA, from a bovine liver library, by complementation of an ATR-deficient Salmonella mutant for color formation on aldehyde indicator medium. Subsequently, sequence similarity searching identified the homologous human cDNA and its corresponding gene. Both the human and bovine cDNAs are shown to express ATR activity and complement an ATR-deficient bacterial mutant. In addition, Western blots were used to show that expression of the human ATR was altered in cell lines derived from three cblB patients compared with a cell line derived from a normal individual. We propose that the human gene identified here coincides with the cblB complementation group, defects in which lead to methylmalonic aciduria. The identification of genes involved in methylmalonic aciduria is important for the development of improved methods for the diagnoses and treatment of this devastating disorder. Materials and Methods Chemicals and Reagents CNCbl and HOCbl were purchased from Sigma Chemical Company (St. Louis, MO). Titanium (III) citrate was prepared as previously described (Bobik and Wolfe
39 1989). 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) and isopropyl--D-thiogalactopyranoside (IPTG) were from Diagnostic Chemicals Ltd. (Charlottetown, Prince Edward Island, Canada). Restriction enzymes and T4 DNA ligase were from New England Biolabs (Beverly, MA). Other chemicals were from Fisher Scientific (Norcross, GA). Bacterial Strains and Growth Media Bacterial strains used in this study are listed in Table 2-1. The minimal medium used was NCE (Vogel and Bonner 1956, Berkowitz et al. 1968) supplemented with 0.4% 1,2-propanediol, 200 ng/ml of CNCbl, 1 mM MgSO4, 0.3 mM each of valine, isoleucine, leucine, and threonine. LB medium was the rich medium used (Difco Laboratories, Detroit, MI) (Miller 1972). MacConkey and aldehyde indicator media were supplemented with 1,2-propanediol and CNCbl and prepared as previously described (Johnson et al. 2001). General Protein and Molecular Methods and ATR Assays Bacterial transformation, polymerase chain reaction (PCR), restriction enzyme digests, and other standard molecular and protein methods were performed as previously described (Maniatis et al. 1982, Johnson et al. 2001). ATR assays were also performed as previously reported (Johnson et al. 2001). P22 Transductions Transductional crosses were performed as described previously (Davis et al. 1980). In preparing lysates of galE mutant strains, overnight cultures were grown on LB medium supplemented with 0.2% galactose and 0.2% glucose with the addition of appropriate antibiotics. A P22 HT105/1 int-201 phage was used for transductional crosses at a concentration of 2x108 phage/ml (Schmieger 1971).
40 Screening the Bovine and Human Liver cDNA Libraries Uni-ZAP XR bovine and human liver cDNA libraries were from Stratagene (La Jolla, CA). Titering, amplification, and mass excision of the cDNA carried on pBlueScript SK(+/-) were done according to the Manufacturerâ€™s protocol except that the titering procedure used F-top agar with 0.2 mM thymine, but no NaCl (Miller 1972) instead of NZY top agar. Following mass excision, the cDNA expression plasmids were purified using a QIAprep spin mini prep kit (Qiagen, Chatsworth, CA). A portion of the resulting expression library was used to transform S. enterica strain BE253 by electroporation. Strain BE253 is ATR-deficient due to pduO and cobA mutations, and carries pSJS1240 which provides rare tRNAs that enhance expression of heterologous genes in S. enterica. Following electroporation, transformation mixtures were suspended in molten aldehyde indicator medium that had been supplemented with 1,2-propanediol, HOCbl and 100 g/ml ampicillin (Amp), and cooled to 45 C. The molten medium was poured into 100 x 15 mm sterile Petri dishes, allowed to solidify, and incubated at 37 C in the dark for ~12 h. Resultant colonies were screened for red/brown color formation. This procedure allowed screening of about 5,000 transformants per plate. Cloning the Bovine Adenosyltransferase Coding Sequence for High-Level Expression PCR was used to amplify the bovine ATR coding sequence. Plasmid pNL121, isolated in this study from a bovine cDNA library, provided the template DNA. The primers used for amplification of the full-length coding sequence were 5â€™-GCCGCCGGTACCGATGACGACGACAAGTTCGGCACGAGCCCGGGAGGT-3â€™ (forward) and 5â€™-GCCGCCAAGCTTGCTTGGTTCCTCGATGAAGCA-3â€™ (reverse). To eliminate the predicted mitochondrial targeting sequence (MTS), forward primer
41 5â€™-GCCGCCGGTACCGATGACGACGACAAGCCCCAGGGCGTGGAAGACGGG-3â€™was used in conjunction with the reverse primer described above. These primers introduced KpnI and HindIII restriction sites into the PCR products and were designed such that following cloning, the bovine ATR coding sequence would be fused to both N-terminal glutathione S-transferase and His6 tags. PCR products obtained using the primers described above were restricted with KpnI and HindIII, and ligated to the pET-41a expression vector (Novagen, Cambridge, MA) that had been similarly digested (Maniatis et al. 1982). Ligation mixtures were used to transform E. coli DH5 by electroporation and transformants were selected by plating on LB-kanamycin (Kan) medium. Pure cultures were prepared from selected transformants and plasmid DNA isolated from these strains was analyzed by restriction digestion and DNA sequencing. Clones having the expected DNA sequences were transformed into E. coli strain BL21 (DE3) RIL (Stratagene) for high-level expression. Cloning the Human Adenosyltransferase Coding Sequence for High-Level Expression The human ATR coding sequence was cloned via PCR using a strategy similar to that described above for the bovine enzyme. IMAGE cDNA clone 2822202 provided the template DNA (Incyte Genomics, Palo Alto, CA). For amplification of the full-length coding sequence, the following primers were used: forward 5â€™-GCCGCCAGATCTGGATGACGACGACAAGATGGCTGTGTGCGGCCTGG-3â€™ and reverse 5â€™-GCCGCCAAGCTTTCAGAGTCCCTCAGACTCGGCCG-3â€™. To eliminate the putative MTS, primer 5â€™-GCCGCCAGATCTGGATGACGACGACAAGC CTCAGGGCGTGGAAGACGGG-3â€™ was used as the forward primer. The primers described above, introduced BglII and HindIII restriction sites that were used for cloning
42 into the pET-41a expression vector. The BglII site was positioned such that the resulting clones would express the human ATRs as fusion proteins with N-terminal GST and His6 tags. Ligation, transformation and analysis of clones were performed as described above for the bovine expression clones. Clones with the expected DNA sequence were transformed into E. coli strain BL21 (DE3) RIL for high-level expression. Cloning the Human Adenosyltransferase Coding Sequence for Complementation Studies For complementation studies, the human ATR coding sequence was amplified by PCR and cloned into plasmid pLAC22 which allows IPTG-inducible expression in S. enterica (Warren et al. 2000). The primers used for amplification were forward, 5â€™-GCCGCCAGATCTTATGCCTCAGGGCGTGGAAGACGGG-3â€™ and reverse, 5â€™-GCCGCCAGCTTTCAGAGTCCCTCAGACTCGGCCG-3â€™. These primers eliminate the putative MTS and provide the needed ATG start triplet such that the first five amino acids of the expressed protein would be MPQGV. The PCR product was restricted with BamHI and HindIII and ligated into pLAC22 that had been digested with BglII and HindIII. Ligation mixtures were used to transform S. enterica strain BE253 (ATR-deficient) by electroporation. Transformants were selected on AIM supplemented with 1,2-propanediol and Amp. Use of this medium allowed the identification of clones expressing ATR activity. Pure cultures were prepared from selected transformants and plasmid DNA isolated from these strains was analyzed by restriction digestion and DNA sequencing. Clones having the expected DNA sequences were moved into strain BE266 (pduO cobA) via P22 transduction. Pure cultures prepared from the resultant colonies were determined to be phage-free by cross-streaking against P22 H5 and used for complementation studies.
43 Growth of Adenosyltransferase Expression Strains and Preparation of Cell Extracts The E. coli strains used for expression of the bovine and human ATRs were grown on LB supplemented with 25 g/ml Kan at 37C with shaking at 275 rpm in a New Brunswick Scientific shaker incubator. Cells were grown to an absorbance of 0.6-0.8 at 600 nm and protein expression was induced by the addition of 1 mM IPTG. Cells were incubated at 37C with shaking at 275 rpm for an additional 3 hours. Cells were removed from the incubator, held on ice for 10 minutes, and then harvested by centrifugation at 6,690 x gmax for 10 minutes using a Beckman JLA-10.500 rotor. The cells were resuspended in 3 ml of 50 mM sodium phosphate, 300 mM NaCl, pH 7, and broken using French Pressure Cell (SLM Aminco, Urbana, IL) at 20,000 psi. Phenylmethylsulfonyl fluoride was added to the cell extract to a concentration of 100 g/ml to inhibit proteases. The crude cell extract was centrifuged at 16,000 rpm (31,000 x gmax) for 30 minutes using a Beckman JA20 rotor to separate the soluble and insoluble fractions. The supernatant was decanted (soluble fraction), and the pellet was resuspended in 1 ml of 50 mM sodium phosphate, 300 mM NaCl, pH 7 (inclusion body fraction). Both fractions were used for the ATR assays. Growth Curves Cells to be used as inocula for growth curves were grown overnight at 37C in 2 ml LB medium or LB medium supplemented with Amp at 100 g/ml for strains carrying pLAC22. A portion of the LB culture (1.5 ml) was pelleted by centrifugation, and resuspended in 1 ml of minimal medium. Then resuspended cells (125 l) were used as the inoculum. Cells were grown in 16 x 150-mm culture tubes containing 5 ml of minimal medium and incubated at 37C in a New Brunswick model C-24 water bath with
44 a shaking speed of 250 rpm. Cell growth was determined by measuring the absorbance at 600 nm using a Milton Roy model Spectronic 20D+ spectrophotometer. Western Blots Human skin fibroblasts from one normal individual (MCH45) and three patients diagnosed with cobalamin b (cblB) disorder (WG1680, WG1879, and WG2127) were obtained from the Repository for Mutant Human Cell Strains at Montreal Childrenâ€™s Hospital. These cell lines were grown in Eagleâ€™s minimum essential medium supplemented with Earleâ€™s salts, L-glutamine, 1% nonessential amino acids, 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, 100 g/ml streptomycin, and 0.2 g/ml HOCbl and incubated at 37C with 5% CO2, 95% air mixture. For lysate preparation, each of the cell lines were cultured in 3, T150 cm2 tissue culture flasks and grown to confluence. Harvested fibroblasts were washed twice with phosphate-buffered saline and lysed in 50 mM Tris, pH 7.3, 100 mM KCl, 5 mM MgCl2, 1.6% Triton-X-100 and a protease inhibitor mixture (Roche Diagnostics, Manheim, Germany). Insoluble cellular matter was removed by centrifugation at 12,000 x g at 4 C for 15 minutes. Protein concentration of the soluble extract was determined using a Micro BCA assay (Pierce, Rockford, IL). Cell Extracts (220 g protein) were fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a Hybond-P, polyvinylidene difluoride membrane (Amersham Biosciences). The membrane was incubated at room temperature for one hour with rocking in 10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05% Tween 20 (TBST) containing 3% bovine serum albumin and 3% mouse serum. The primary antibody used was affinity-purified rabbit IgG anti-human-ATR diluted 1:1000. The secondary antibody was monoclonal
45 anti-rabbit IgG (-chain specific) biotin conjugate (Sigma) and was used at a 1:25,000 dilution. All membrane incubations were carried out at room temperature for one hour with rocking in TBST. The membrane was prepared for detection using streptavidin horseradish peroxidase conjugate (Caltag Laboratories, Burlingame, CA) diluted 1:10,000, and Immun-Star HRP Chemiluminescent Kit (Bio-Rad, Hercules, CA) following the Manufacturerâ€™s protocol. Membranes were exposed to X-ray film from Research Products International (Mt. Prospect, IL). DNA Sequencing and Analysis DNA sequencing was carried out by University of Florida, Interdisciplinary Center for Biotechnology Research, DNA Sequencing Core facility, and University of Florida, Department of Microbiology and Cell Science, DNA Sequencing Facility as described previously (Bobik et al. 1999). Sequence similarity searches were carried out using the Blast family of sequence analysis software (Altschul et al. 1990, Altschul et al. 1997). Multiple sequence alignments were done with ClustalX using default parameters except as noted below (Thompson et al. 1997). The slow and accurate method was used for pairwise alignments with a gap opening and extension penalties of 35 and 0.75, respectively. Multiple sequence alignment parameters were gap opening and extension penalties of 15 and 0.3, respectively, and delay divergent sequences were set at 25%. Results Screening of the Bovine and Human Liver cDNA Libraries for Clones that Express Adenosyltransferase Activity Our previous studies showed that S. enterica strain BE253 forms red/brown colonies on AIM supplemented with 1,2-propanediol and CNCbl when it is transformed with an ATR expression plasmid (Johnson et al. 2001). The basis of the color formation
46 is that ATR activity restores the ability of strain BE253 to convert CNCbl to AdoCbl, the required cofactor for diol dehydratase. Given AdoCbl, diol dehydratase converts 1,2-propanediol to propionaldehyde which reacts with components in the aldehyde indicator medium to form a red/brown precipitate (Johnson et al. 2001). Hence, we expected that BE253 would be useful for screening expression libraries for ATR activity. Accordingly, we attempted to identify a human ATR cDNA by transforming strain BE253 with a human liver cDNA expression library and screening the resultant colonies for red/brown color formation on AIM. Approximately 250,000 human cDNAs were screened, but no red/brown colonies were found. Subsequently, a bovine liver cDNA library was similarly screened and four positive clones were isolated. The DNA sequences of the four clones were determined and found to be identical. The clone was 1058 bp in length, and included a poly(A) tail indicating that its 3' end was complete. However, analyses indicated that the 5' end of the clone was incomplete. The clone included a 228-amino acid open reading frame that would be expressed as an N-terminal -galactosidase fusion protein (as was expected given the cloning method employed), but it lacked the expected ATG triplet. Hence, the bovine clone apparently lacked a portion of its 5' end. After the DNA sequence of the bovine cDNA clones was determined, Blastp searches of the NCBI nonredundant database were conducted, and these searches showed that the 228-amino acid protein encoded by the bovine cDNA clone was 29% identical (Expect = 8 x 10-15) to the N-terminal domain (165 amino acids) of the PduO ATR of S. enterica (Johnson et al. 2001). The findings that the bovine cDNA isolated both complemented an ATR-deficient mutant of S. enterica and encoded a protein with
47 sequence similarity to a known ATR enzyme suggested that the bovine clone also encoded an ATR enzyme. Identification of a Human Gene Related to the Bovine Adenosyltransferase cDNA Blastp searches showed that the putative bovine ATR enzyme was 88% identical to a human protein encoded by IMAGE cDNA clone 2822202 (accession number BC005054). The human cDNA clone was 1128 bp in length and included an appropriate ATG triplet and a poly(A) tail indicating that it was a full-length clone. Further sequence analyses showed that the predicted protein encoded by the human cDNA was 250 amino acids in length and was homologous over its entire length to the putative bovine ATR except that it included 19 additional N-terminal amino acids (Figure 2-1). This suggested that the bovine clone was nearly full-length, lacking only a portion of its N-terminus, probably a small region of its MTS (see below). Perhaps more importantly, however, the high identity between the human and bovine sequences indicated that both encoded proteins with similar functions. Furthermore, sequence similarity searches showed human cDNA BC005054 encoded a protein with 26% amino acid identity the PduO ATR of S. enterica. Thus, these results tentatively identified sequence BC005054 as the human ATR cDNA. Subsequent sequence analyses showed human cDNA BC005054 was 99% identical to nine regions of human chromosome XII. The IMAGE clone BC005054 encoded a protein with 2 amino acid substitutions compared with the putative protein encoded by chromosome XII (Arg-19 to Gln and Met-239 M to Lys). These changes may represent natural polymorphisms or may have resulted from mutations introduced during cDNA preparation. The nine regions on chromosome XII were the only regions of the human genome that showed significant homology to human cDNA BC005054. Hence, it seems
48 likely that these regions correspond to nine exons that encode the human ATR enzyme. The exon structure of the human ATR is indicated in Figure 2-1. Putative Subcellular Localization of Adenosyltransferase Enzymes The amino acid sequences of the human and bovine ATR enzymes were aligned with 20 related sequences obtained from GenBank (Figure 2-1). This alignment showed that the human, bovine, and mouse ATR sequences included about 50-60 additional N-terminal amino acids compared with their prokaryotic homologues. In higher organisms, ATRs localize to mitochondria (Banerjee and Chowdhury 1999); hence, the additional N-terminal amino acids of the eukaryotic sequences might represent MTSs. Prediction of the subcellular localization of the human and mouse proteins using Predotar (Small et al. 2004) and MitoProt II (Claros and Vincens 1996) software indicated mitochondrial localization: human, MitoProt and Predotar scores both = 0.89; and mouse MitoProt and Predotar scores = 0.97 and 0.99, respectively. Because the bovine clones were apparently partial sequences, reliable prediction of subcellular localization of the presumptive bovine ATR enzyme was not possible (Claros and Vincens 1996, Small et al. 2004). High-Level Expression of the Bovine and Human Adenosyltransferases in E. coli E. coli strains were constructed for high-level expression of the presumptive bovine and human ATR enzymes. Clones were constructed to allow expression of these enzymes with and without sequences presumed to be involved in mitochondrial targeting. Our concern was that the MTS sequences might interfere with enzyme activity as such sequences are normally removed during mitochondrial localization (Paschen and Neupert 2001). The arrows in Figure 2-1 indicate the regions of the bovine and human ATRs that were expressed by clones that eliminated their MTSs. Furthermore, in all cases, the
49 ATRs were produced as fusion proteins with N-terminal glutathione S-transferase and His6 tags that could be removed by enterokinase cleavage if desired. Production of the presumptive bovine and human ATR fusion proteins was monitored by SDS-PAGE (Figure 2-2). Both the soluble (lanes 2 thru3) and insoluble fractions (lanes 5-7) were analyzed. Strains BE255 and BE256 produced large amounts of protein with molecular masses of 55 and 52 kDa, respectively (Figure 2-2 A, lanes 3, 4, 6, and 7). These values are near the predicted masses (58 and 54 kDa) for the bovine ATR fusion proteins with and without the presumptive MTS and including the expression tags. In contrast, a strain carrying the expression plasmid without insert (BE237) produced little protein near these molecular masses (Figure 2-2 A, lanes 2 and 5) indicating that BE255 and BE256 were indeed producing large amounts of bovine ATR fusion proteins with and without the presumptive MTS. Expression of the human ATR fusion proteins was also monitored by SDS-PAGE (Figure 2-2 B). The predicted molecular masses of the human ATR fusion proteins with the expression tags, with and without the predicted MTS are 58 and 55 kDa, respectively. As shown in Figure 2-2 B, large amounts of proteins with calculated molecular masses of 56 and 52 kDa were produced by expression strains BE257 (ATR + MTS) and BE258 (ATR without MTS), (Figure 2-2 B, lanes 3, 4, 6, and 7) but not by the control strain BE237 (lanes 2 and 5) which carried that expression plasmid without insert. Assay of the Bovine and Human Adenosyltransferase Fusion Proteins for ATR Activity The cell extracts analyzed by SDS-PAGE (Figure 2-2) were tested for ATP:cob(I)alamin ATR activity (Table 2-2). Substantial activity was found in both soluble and inclusion body extracts from strains BE255 (bovine ATR + MTS), BE256
50 (bovine ATR), BE257 (human ATR + MTS), and BE258 (human ATR). Of the four bovine ATR extracts tested, the highest specific activity measured was 85.7 nmol min-1 mg-1 found in the inclusion body fraction from strain BE255 (ATR + MTS). The highest specific activity found in the human ATR extracts tested was 98 nmol min-1 mg-1 in the inclusion body fraction containing human ATR without its presumptive MTS. For each ATR fusion protein, the majority of the activity was found in the soluble fraction (Table 2-2, column 4). For both the human and bovine cell extracts, ATR activity was linear with protein concentration (data not show). Furthermore, when the substrates, ATP and/or cob(I)alamin were excluded from the assay mixture, no activity was detected. Also, as expected, cell extracts from strain BE237 (T7 expression vector without insert) expressed no measurable ATR activity. Complementation of an S. enterica Mutant Deficient in ATR Activity by a Human Adenosyltransferase cDNA Clone We previously showed that an S. enterica strain deficient for both the CobA and PduO ATRs (cobA pduO) was unable to grow on 1,2-propanediol due to an ATR deficiency (Johnson et al. 2001). To test whether the human ATR cDNA could complement this defect, we compared the growth of the wild-type strain, BE265 (pduO cobA /pLAC22-human ATR) and BE263 (pduO cobA /pLAC22-no insert) on 1,2-propanediol minimal medium (Figure 2-3). The doubling times of the wild-type and strain BE265 (pduO cobA /pLAC22-human ATR cDNA) were found to be comparable; they were 8.4 and 8.9 h, respectively. Control experiments showed that strain BE263, which carried the pLAC22 vector without insert, grew minimally on 1,2-propanediol, and that growth of strain BE265 (pduO cobA/pNL135-human ATR) required the addition of IPTG as was expected since expression of the human ATR in this strain is under control
51 of the lacI repressor (not shown). Thus, clearly, the human ATR cDNA complemented the ATR-deficient bacterial mutant for AdoCbl-dependent growth on 1,2-propanediol. These results provided further evidence that human cDNA BC005054 encodes an ATR enzyme, and also showed that this enzyme can function as an ATR under physiological conditions albeit in a heterologous host. Level of Human Adenosyltransferase Enzyme in Normal and cblB Mutant Human Skin Fibroblasts Above, we presented biochemical and genetic evidence that human cDNA BC005054 encodes an ATR enzyme. If defects in this enzyme underlie cblB methylmalonic aciduria, its expression or stability might be altered in cblB mutant cell lines. To examine the expression of this enzyme in normal and cblB mutant human skin fibroblasts, Western blots were performed on lysates from these cells using antibody against recombinant human ATR described above. Figure 2-4 shows a representative experiment. Lane 5 contained recombinant human ATR lacking both the mitochondrial targeting sequence and the expression tags. In this lane, a single protein band with an approximate molecular mass of 25 kDa was recognized by the anti-ATR antibody as expected. In lane 1, which contained cell extracts from normal skin fibroblasts, the anti-human ATR antibodies recognized two bands. One of these bands was similar in molecular mass to the recombinant human ATR (approximately 25 kDa) and the second band was of lower mass (approximately 23 kDa). Neither the 25 kDa nor the 23 kDa band was observed in blots performed with pre-immune serum (data not shown) indicating that these bands most likely represent two processed forms of the human ATR. Interestingly, ATR-reactive polypeptides of these molecular masses were differentially expressed in the three cblB cell lines tested (lanes 2-3). The cblB mutants WG1680 and
52 WG2127 did not express detectable levels of the 25 kDa form of the ATR and expressed reduced levels of the 23 kDa form (lanes 2 and 4). For cblB mutant WG1879, the 25 kDa form was reduced, and the 23 kDa form was not detectable. These results indicated that expression of the ATR was significantly altered in cblB mutant human skin fibroblasts. These results provide direct evidence that defects in the ATR identified in this study underlie cblB methylmalonic aciduria. Conserved Amino Acids and Distribution of Adenosyltransferase Enzymes The human and bovine ATR enzymes were aligned with 20 related sequences obtained from GenBank (Figure 2-1). Of the sequences shown, the function of the H. sapiens, B. taurus, and S. enterica ATRs is supported by biochemical evidence presented here and previously (Johnson et al. 2001). To obtain additional information about the function of the remaining prokaryotic ATRs, we examined their genomic context. Genes encoding ATR homologues from S. enterica, L. innocua, L. monocytogenes, B. halodurans, C. perfringes, C. pasteurianum, and K. pneumonia were found proximal to either AdoCbl-dependent enzymes or AdoCbl biosynthetic genes. Since bacteria frequently cluster genes of related function, these finding suggest that the genes from these organisms do indeed encode ATR enzymes. It is also notable that there are several regions of high conservation in all the ATRs aligned suggesting that many are indeed ATR enzymes (Figure 2-1). Thus, PduO-type ATR enzymes appear to have broad phylogenetic distribution, being found in mammals, gram positive and gram negative bacteria, and the archaea. The highly conserved regions found in the ATRs also indicate amino acids likely to be essential for enzymatic activity, and may represent sites directly involved in catalysis or in binding of substrates, ATP and cob(I)alamin. There are at least two different ways
53 in which proteins bind B12, the "base-on" and "base-off" modes. Base-off binding involves displacement of the lower axial ligand of B12 (dimethylbenzimidazole) with an imidazole side-chain of a histidine residue (Drennan et al. 1994, Shibata et al. 1999, Marsh and Drennan 2001). The sequence containing the coordinating histidine (D-X-H-X-X-G), which is conserved among proteins that bind B12 in the â€œbase-offâ€ mode, is absent from PduO-type ATRs examined in this study. This suggests that the ATR enzymes described in this study bind B12 in the â€œbase-onâ€ mode. Discussion In this report we identified human and bovine cDNAs that encode ATR enzymes. The highest specific activities measured in cell extracts from expression strains were 85.7 nmol min-1 mg-1 protein for the bovine ATR and 98 nmol min-1 mg-1 protein for the human ATR. These values are comparable to those previously reported for purified CobA ATR, and partially purified PduO ATR which were 53 and 312 nmol min-1mg-1 protein, respectively (Suh and Escalante-Semerena 1993, Suh and Escalante-Semerena 1995). The bovine ATR cDNA was isolated based on its ability to complement an ATR-deficient S. enterica mutant for color formation on indicator medium, and the human ATR cDNA was shown to restore the ability of an ATR-deficient mutant to grow on 1,2-propanediol in an AdoCbl-dependent fashion. This demonstrated that the human and bovine cDNAs described here encode ATR enzymes that are active under physiological conditions, although in a heterologous host. Moreover, the bovine and human ATR cDNAs had 29% and 26% identity to the PduO ATR of S. enterica (Johnson et al. 2001) and the human enzyme included a presumptive MTS as expected (Fenton et al. 2000). Thus, genetic, biochemical, and bioinformatic evidence indicate that the bovine and human cDNAs analyzed here encode ATR enzymes.
54 Prior studies have indicated that ATR defects in humans lead to methylmalonic aciduria (Fenton and Rosenberg 1981, Banerjee and Chowdhury 1999). MCM is known to require AdoCbl for activity (Banerjee and Chowdhury 1999) and fibroblast extracts from methylmalonic aciduria patients with defects in the cblB complementation group have been shown to lack ATR activity (Fenton and Rosenberg 1981). The ATR cDNA identified in this study corresponded to a single human gene comprised of nine exons found on chromosome XII. Furthermore, Western blots indicated that expression of this ATR was altered in cblB mutant human cell lines compared to normal cell lines. Thus, we propose that the human gene identified here corresponds to the cblB complementation group that is involved in methylmalonic aciduria. The approach used in this study to identify the human ATR cDNA involved screening mammalian cDNA expression libraries for clones that complemented an ATR-deficient bacterial mutant. This general strategy may have application to the identification of additional cDNAs involved in cobalamin metabolism. Human cDNAs encoding the -ligand transferase and the cob(III)alamin and cob(II)alamin reductases (the cblACD complementation groups) have not yet been identified (Rosenblatt and Fenton 1999, Watkins and Rosenblatt 2001). In this regard, it is notable that in this study the human ATR cDNA was not directly isolated, but rather the bovine ATR cDNA was isolated and sequence similarity searching was used to identify the homologous human gene. The fact that our screen allowed direct isolation of a bovine ATR cDNA, but not a human ATR cDNA may reflect the relative abundance of ATR mRNA in the human versus the bovine liver. Ruminant livers have among the highest levels of B12-dependent enzymes measured in any tissue (Schneider and Stroinski 1987). Thus, ruminant liver
55 libraries may be particularly useful for isolating mammalian cDNAs involved in cobalamin metabolism. Importantly, the identification of human genes involved in methylmalonic aciduria should help with the development of improved methods for diagnosis and treatment of this rare but devastating disease. Knowledge of the relevant genes will allow DNA-based methods of diagnosis following amniocentesis or chorionic villi sampling. Such techniques will allow the identification of the specific genetic lesions involved, and this may help guide treatment. Some cases of methylmalonic aciduria respond to high-dose B12 therapy and it seems likely that such cases will be associated with specific mutations (Ampola et al. 1975, Matsui et al. 1983, Zass et al. 1995). Moreover, as the needed methodologies become available, knowledge of the genes involved in cobalamin metabolism may also help with the development of gene or enzyme therapies as treatments for methylmalonic aciduria.
56 Table 2-1. Bacterial strains Species Strain Genotype E. coli BL21 (DE3) RIL (E. coli B) F-ompT hsdS (rB-mB-) dcm+ Tetr gal (DE3) endA Hte (argU ileY leuW Camr) SOLRTM (Stratagene) e14-(McrA-) (mcrCB-hsdSMR-mrr)171 sbcC recB recJ uvrC umuC::Tn5 (Kanr) lac gyrA96 relA1 thi-1 endA1 R [Fâ€™ proAB lacIqZM15]c Su(nonsuppressing) XL1-Blue MRFâ€™ (Stratagene) D(mcrA)183 (mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [Fâ€™ proAB lacIqZM15 Tn10 (Tetr)]c BE237 BL21 (DE3) RIL/pET-41a (T7 expression vector without insert) BE255 BL21 (DE3) RIL/pNL128 (bovine ATR+MTS) BE256 BL21 (DE3) RIL/pNL129 (bovine ATR without MTS) BE257 BL21 (DE3) RIL/pNL132 (human ATR+MTS) BE258 BL21 (DE3) RIL/pNL133 (human ATR without MTS) S. enterica serovar Typhimurium LT2 TR6579 metA22 metE551 trpD2 ilv-452 hsdLt6 hsdSA29 HsdBstrA120 GalE-Leu-ProBE253 TR6579 cobA::dcam pduO651 yeex::kanr/pSJS1240 (ileX, argU) BE254 BE253/pNL121 (pBlueScript with bovine ATR) BE266 pduO651 cobA366::Tn10dCam/pSJS1240 (ileX, argU) BE263 pduO651 cobA366::Tn10dCam/pLAC22/pSJS1240 (ileX, argU) BE265 pduO651 cobA366::Tn10dCam/pNL135 (human ATR without MTS under plac control)
57 Figure 2-1. Multiple sequence alignment of adenosyltransferase enzymes. ClustalX was used to align the prokaryotic, eukaryotic, and archaeal homologues of ATR enzymes. Identical residues are shaded black, and highly conserved residues are shaded gray. The predicted mitochondrial targeting sequence determined by MitoProtII software is underlined in the human sequence. Triangles separate the human ATR into nine regions that correspond to exons on chromosome XII. Arrows represent the PCR primers used for amplification of the human ATR without its presumptive MTS.
58 116.25A.97.44566.221.53114.4 B.97.44566.221.531kDa kDa 12345671234567 116.25A.97.44566.221.53114.4 B.97.44566.221.531kDa kDa 1234567123456712345671234567 Figure 2-2. SDS-PAGE analysis of cell extracts from bovine (A) and human (B) ATR expression strains. Panels A and B lane 1, molecular mass markers. Panel A, lanes 2 thru 4 are soluble extracts of BE237 (T7 expression vector without insert), BE255 (bovine ATR + MTS), and BE256 (bovine ATR without MTS). Panel A, lanes 5 thru 7 are inclusion body fractions from strains BE237, BE255, and BE256. Panel B, lanes 2 thru 4 are soluble extracts from BE237 (T7 expression vector without insert), BE257 (human ATR + MTS), and BE258 (human ATR without MTS), respectively. Lanes 5 thru 7 are inclusion body fractions from strains BE237, BE257, and BE258.
59 Table 2-2. Specific activities of bovine and human ATP:cob(I)alamin ATRs Strain S/I* Total activity nmol min-1 Total activity % Specific activity nmol min-1 mg-1 protein S 2021 95 47 BE255 (bovine ATR+MTS) I 107 5 85.7 S 3364 84 44.1 BE256 (bovine ATR without MTS) I 632 16 30 S 2445 52 37.5 BE257 (human ATR+MTS) I 2296 48 60.8 S 5481 57 63 BE258 (human ATR without MTS) I 4144 43 98 *S=soluble fraction, I=inclusion body fraction
60 0.101020304 0 1.00.5Time (hours)OD600 0.101020304 0 1.00.5Time (hours)OD600 Figure 2-3. Complementation of an ATR-deficient bacterial mutant for AdoCbl-dependent growth on 1,2-propanediol by a plasmid that expresses the human ATR. Cells were grown in minimal 1,2-propanediol medium supplemented with HOCbl and 0.1 mM IPTG. Growth was determined by measuring the absorbance of cultures at 600 nm (OD600), and data are shown as a semi-log plot. , Wild-type (S. enterica serovar Typhimurium LT2); , BE265, ATR-deficient/pNL135(human ATR under plac control); , BE263, ATR-deficient /pLAC22 (vector without insert).
61 1234525 kDa 1234525 kDa Figure 2-4. Western blot analysis of ATR expression by normal and cblB mutant cell lines. Extracts were prepared from cultured fibroblast lines obtained from a normal individual and three cblB patients. Lane 1: cell extract from the MCH45 control cell line. Lanes 2 thru 4: cell extracts from cblB mutant cell lines WG1680, WG1879 and WG2127, respectively. 220 g of protein was loaded into each of lanes 1 thru 4 (It was necessary to load relatively high amounts of protein because of the low abundance of the ATR enzyme in skin fibroblasts). Lane 5 contained 100 pg recombinant human ATR lacking the mitochondrial targeting sequence and affinity tags. The arrow shows the location of the recombinant ATR enzyme (approximately 25 kDa). The experiment was performed twice with similar results.
CHAPTER 3 PURIFICATION AND INITIAL CHARACTERIZATION OF THE HUMAN ATP:COB(I)ALAMIN ADENOSYLTRANSFERASE AND ITS INTERACTION WITH METHIONINE SYNTHASE REDUCTASE Introduction The vitamin B12 coenzymes, adenosylcobalamin (AdoCbl) and methylcobalamin (CH3Cbl) are required cofactors for at least fifteen different enzymes that have a broad but uneven distribution among living forms (Schneider and Stroinski 1987, Banerjee 1999). In humans, two B12-dependent enzymes are known (Kolhouse and Allen 1977, Mellman et al. 1977). AdoCbl -dependent methylmalonyl-CoA mutase (MCM) catalyzes the reversible rearrangement of methylmalonyl-CoA to succinyl-CoA (Banerjee and Chowdhury 1999). MCM is found in the mitochondrial matrix and it is required for the complete catabolism of compounds degraded via propionyl-CoA including branched-chain amino acids, odd-chain fatty acids, and cholesterol (Figure 3-1) (Banerjee and Chowdhury 1999). The second B12-dependent enzyme known in humans is CH3Cbl-dependent methionine synthase (MS). This enzyme is found in the cytoplasm where it catalyzes the conversion of methyltetrahydrofolate and homocysteine to tetrahydrofolate and methionine (Matthews 1999). In mammals, methionine is an essential amino acid, and MS plays a role in recycling the S-adenosylhomocysteine formed from S-adenosylmethionine-dependent methylation reactions (Weissbach et al. 1963). Humans are incapable of synthesizing AdoCbl and CH3Cbl de novo and are dependent on dietary sources of complex cobalamin precursors, such as vitamin B12 62
63 (cyanocobalamin, CNCbl) and hydroxycobalamin (HOCbl) (Stabler 1999). The pathway by which these precursors are metabolized to the coenzyme forms is thought to be similar in both prokaryotes and eukaryotes (Figure 3-1). For AdoCbl synthesis, CNCbl is proposed to be converted to glutathionylcobalamin (GSCbl), reduced successively to cob(II)alamin and cob(I)alamin respectively and finally, adenosylated to AdoCbl (Figure 3-1) (Friedmann 1975, Huennekens et al. 1982, Pezacka et al. 1990, Pezacka 1993, Watanabe et al. 1996). For the synthesis of CH3Cbl, cob(II)alamin associated with MS, is reductively methylated to form CH3Cbl (Figure 3-1) (Drummond and Matthews 1994, Matthews 1999). For this reaction (which is also used for the reductive activation of MS following adventitious oxidation of the cobalamin cofactor), S-adenosylmethionine is the methyl-group donor and methionine synthase reductase (MSR) reduces cob(II)alamin to cob(I)alamin (Leclerc et al. 1998, Olteanu and Banerjee 2001). In humans, inherited defects in the MCM or MS structural genes or in the genes needed for the synthesis of B12 coenzymes result in methylmalonic aciduria, homocystinuria, or combined disease. These rare disorders, which are often fatal in the first year of life, result from recessive autosomal mutations that fall into nine complementation groups (mut, cblABCDEFGH) (Qureshi et al. 1994, Kapadia 1995, Rosenblatt and Fenton 1999, Watkins and Rosenblatt 2001). The cblF, cblC and cblD defects lead to combined disease. Prior studies indicated that cblF mutations affect cobalamin transport from the lysosome to the cytoplasm, while cblC and cblD mutations impair the conversion of complex precursors to cob(II)alamin (Mellman et al. 1979, Watkins and Rosenblatt 2001). The cblE and cblG complementation groups correspond
64 to the genes for MSR (MTRR) and MS (MTR), respectively, and defects in either gene results in homocystinuria (Cooper and Rosenblatt 1987, Watkins and Rosenblatt 2001). The mut group corresponds to the gene that encodes MCM (MUT). The cblA group (MMAA gene) encodes a protein proposed to protect MCM from inactivation (Korotkova and Lidstrom 2004) and recent studies have shown that the cblB complementation group (MMAB gene) encodes a mitochondrial ATP cob(I)alamin adenosyltransferase (ATR) (Dobson et al. 2002, Leal et al. 2003). Mutations in the cblH complementation group also result in methylmalonic aciduria, but the nature of the underlying defect and the corresponding gene are unknown (Watkins and Rosenblatt 2001). The human enzyme that catalyzes the reduction of cob(II)alamin to cob(I)alamin for AdoCbl synthesis has not been identified. Recent studies showed that MSR mediates cob(II)alamin reduction for MS activation (Leclerc et al. 1999, Olteanu and Banerjee 2001), raising the possibility that this enzyme might play a role in AdoCbl synthesis. Here we describe the purification and initial biochemical characterization of the human ATR from recombinant E. coli and demonstrate that in vitro human MSR can reduce cob(II)alamin to cob(I)alamin for AdoCbl synthesis by the ATR enzyme. Materials and Methods Chemicals and Reagents Restriction enzymes and T4 DNA ligase were from New England Biolabs (Beverly, MA). Titanium III citrate was prepared as previously described (Bobik and Wolfe 1989). AdoCbl, HOCbl, -nicotinamide adenine dinucleotide phosphate reduced form (NADPH), iodoacetic acid (IA), flavin adenine dinucleotide (FAD), and bovine serum albumin (BSA) were purchased from Sigma Chemical Company (St. Louis, MO). Isopropyl--D-thiogalactopyranoside (IPTG), ultra-pure ammonium sulfate, adenosine
65 5â€™triphosphate (ATP), and dithiothreitol (DTT) were purchased from ICN Biomedicals, Inc. (Aurora, Ohio). Pefabloc SC PLUS was from Roche Diagnostics (Penzberg, Germany). All other chemicals and reagents were from Fisher Scientific (Norcross, GA). Bacterial Strains and Growth Media The bacterial strains used were E. coli DH5 and BL21 (DE3) RIL (Stratagene, La Jolla, CA). LB was the rich medium used (Difco, Detroit, MI) (Miller 1972). LB was supplemented with 25 g/ml kanamycin and 20 g/ml chloramphenicol or as indicated. General Molecular and Protein Methods Agarose gel electrophoresis and restriction enzyme digests were performed using standard protocols (Maniatis et al. 1982, Johnson et al. 2001). PCR products and plasmid DNA were gel purified using QIAquick gel extraction kit (QIAGEN, Inc., Valencia, CA). DNA ligation was accomplished using T4 DNA ligase according to manufacturerâ€™s instructions. Bacterial transformation and SDS-PAGE were performed as previously described (Johnson et al. 2001). ATP:cob(I)alamin Adenosyltransferase Assays ATR assays were performed as previously reported with some modifications (Johnson et al. 2001). Reaction mixtures contained: 200 mM Tris-HCl (pH 8.0), 2.8 mM MgCl2, 10 mM KCl, 0.05 mM HOCbl, 0.4 mM ATP, and 1 mM titanium(III) citrate in a total volume of 2 ml. Reaction components (except for the ATR) were dispensed into cuvettes inside an anaerobic chamber (Coy Laboratories, Ann Arbor, MI). The cuvettes were sealed, removed from the chamber, and incubated at 37C for 2 minutes. Reactions were initiated by addition of purified recombinant ATR, and AdoCbl formation was measured by following the decrease in absorbance at 388 nm ( 388 = 24.9 cm-1 mM-1).
66 Construction of ATR Expression Strains Plasmid pNL166 (Leal et al. 2003) was restricted with BglII and HindIII releasing a 645 bp fragment that encodes the ATR enzyme with a lysine at position 239 (ATR 239K). This fragment was gel purified and ligated into a modified pET-41a expression vector, pTA925 (Johnson et al. 2001). Ligation mixtures were used to transform E. coli DH5 by electroporation and transformants were selected by plating on LB kanamycin medium. Pure cultures were obtained from selected transformants and plasmid DNA isolated from these strains was analyzed by restriction digestion and DNA sequencing. Clones with the expected DNA sequence were transformed into E. coli strain BL21 (DE3) RIL (Stratagene) for high-level protein production. PCR was used to construct the ATR polymorphic variant with a methionine at position 239 (ATR 239M). Plasmid pNL166 (Leal et al. 2003) provided the template DNA. The primers used for amplification were 5â€™-GCCGCCAGATCTTATGCCTCAGGGCGTGGAAGACGGG-3â€™ (forward) and 5â€™-GCCGCCAAGCTTTCAGAGTCCCTCAGACTCGGCCGATGGGTCATTTTTCATGTATATTTTCTCTTGATTCCC-3â€™ (reverse). These primers introduced BglII and HindIII restriction sites into the PCR product and were designed to change the lysine at position 239 to a methionine. Ligation, transformation, and analysis of plasmid DNA were performed as described above. Clones with the expected DNA sequence were transformed into E. coli BL21 (DE3) RIL for protein production. Purification of the Human ATR Variants Soluble cell extracts of the E. coli expression strains were used for the purification of both ATR variants. The growth of the cells and the preparation of cell extracts were carried out as described (Johnson et al. 2001). For ammonium sulfate precipitation, cell
67 extracts of ATR 239K (160 mg protein) and 239M (180 mg protein) were diluted to 1 mg/ml in 50 mM sodium phosphate (pH 7.0), 300 mM NaCl. Then, ultra-pure ammonium sulfate was added as a fine powder with stirring. Precipitated proteins were centrifuged for 15 min at 12,000 x gmax using a Beckman JLA-10.500 rotor. The protein pellets were resuspended in 4 ml of 5 mM potassium phosphate (pH 6.8), and passed through a 0.45 m filter. ATR 239K (60 mg) and 239M (35 mg) were applied to a 60 ml ceramic hydroxyapatite column (Bio-Rad) equilibrated with 5 mM potassium phosphate (pH 6.8). The column was washed with 60 ml equilibration buffer and eluted with a 600 ml linear gradient of 5 mM to 200 mM potassium phosphate (pH 6.8) at a flow rate of 5 ml/min. Fractions containing ATR of the highest purity were pooled and exchanged into 10 mM potassium phosphate (pH 7.0), 50 mM KCl using a Vivaspin centrifugal concentrator with a molecular weight cutoff of 10,000 Daltons (Viva Science, Binbrook, UK). Samples were filtered through a 0.45 m pore-size membrane and further purified using a Mono Q HR 10/10 anion exchange column (Amersham Pharmacia Biotech, Piscataway, NJ). ATR 239K (10 mg) and 239M (9 mg) were applied to the column which had been equilibrated with 10 mM potassium phosphate (pH 7.0), 50 mM KCl. Then, the column was washed with 8 ml equilibration buffer and eluted with a 160 ml linear gradient from 50 to 1000 mM KCl in 10 mM potassium phosphate (pH 7.0) at a flow rate of 4 ml/min. Protein elution was monitored by following the absorbance of the column effluent at 280 nm and 8 ml fractions were collected. Purified ATR was concentrated and stored in 10 mM phosphate buffer (pH 7.0), 130 mM KCl, 50% glycerol at -20C.
68 MSR-ATR Assay The basis of this assay is that MSR converts cob(II)alamin to cob(I)alamin which is in turn converted to AdoCbl by the ATR enzyme. The conversion of cob(I)alamin to AdoCbl proceeds with an increase in absorbance at 525 nm that allows quantitation (525 = 4.8 cm-1 mM-1). Assays contained 200 mM Tris (pH 8.0), 1.6 mM potassium phosphate, 2.8 mM MgCl2, 100 mM KCl, 0.1 mM cob(II)alamin, 0.4 mM ATP, 1 mM DTT, 1 mM NADPH, purified MSR and ATR as indicated in the text. The total volume was 2 ml and anaerobic procedures were used as described for the ATR assay. Cob(II)alamin was prepared by exposing an anoxic solution of 1 mM AdoCbl to a 150 watt incandescent light at a distance of 15 cm for 30 minutes (Johnson et al. 2001). ATR 239K was purified as described above and MSR was purified as described previously (Olteanu and Banerjee 2001). Reaction mixtures were incubated at 37C for 2 min prior to initiating reactions by the addition of an anoxic solution of NADPH. Measurement of Cob(I)alamin with Iodoacetate Iodoacetate reacts rapidly and quantitatively with cob(I)alamin to form carboxymethylcobalamin (CMCbl). This reaction proceeds with an increase in absorbance at 525 nm (525 = 5.1 cm-1 mM-1) and hence provides a facile method for quantitation of cob(I)alamin (Debussche et al. 1991). Anoxic stock solutions of iodoacetate (40 mM) were prepared and used the same day. The stock solution was shielded from light using aluminum foil and was added to assay mixtures just prior to the initiation of reactions. Separation and Quantification of Cobalamins by HPLC HOCbl, AdoCbl, and CMCbl were separated and quantified by HPLC using a NovaPak C18 column (3.9 mm) equipped with a C18 Sentry guard column (Waters,
69 Milford, MA). Samples (200 l) were loaded onto the column and eluted with 30 ml linear gradient of 10 to 90% methanol in 50 mM sodium acetate (pH 4.6) at a flow rate of 1 ml/min. The absorbance of column effluent was monitored at 365 nm, and analytes were quantified by comparison of peak areas to a standard curve. The CMCbl standard was prepared by incubating 2 ml of 200 mM Tris (pH 8.0), 1.6 mM potassium phosphate, 2.8 mM MgCl2, 100 mM KCl, 0.1 mM cob(II)alamin, 1 mM DTT, 0.1 mM FAD, and 0.4 mM iodoacetate at 37C for 1 hour which resulted in quantitative conversion of cob(II)alamin to CMCbl (data not shown). Standard curves were prepared using 5, 10, 20, 30, and 40 nmoles of CMCbl, AdoCbl, or HOCbl. Peak areas were integrated using Waters Breeze software. The standard curves for CMCbl, AdoCbl, and HOCbl, had regression coefficients (R2) of 0.9985, 0.9993, and 0.9956, respectively. Solutions that contained CMCbl or AdoCbl were shielded from light to prevent photolysis. Effect of Ionic Strength on the MSR-ATR System The ionic strength of MSR-ATR assays was varied by the addition of KCl. Ionic strength was calculated using the formula I = 1/2(ciz)2 where I is the ionic strength, ci is the molar concentration of each type of ion, and z is the charge of each ion (Segel 1976). For lower values of ionic strength, it was necessary to reduce the Tris-HCl concentrations. DNA Sequencing DNA sequencing was carried out by University of Florida, Interdisciplinary Center for Biotechnology Research, DNA Sequencing Core facility and University of Florida, Department of Microbiology and Cell Science, DNA Sequencing facility as described previously (Bobik et al. 1999).
70 Results Purification of the Human ATR Variants The human ATR catalyzes the transfer of a 5'-deoxyadenosyl group from ATP to the central cobalt atom of cob(I)alamin to form AdoCbl (Chapter 2, Leal et al. 2003). Two common polymorphic variants of the human ATR having either lysine or methionine as amino acid 239 (ATR 239M and 239K) were recently shown to be present in 54% and 46% of 72 control cell lines, respectively (Dobson et al. 2002). We previously produced ATR 239K as a GST-fusion protein in E. coli (Leal et al. 2003). Here, subcloning and site-directed mutagenesis were used to construct E. coli expression strains that produce high levels of ATR 239K and 239M without fusion tags and lacking their predicted mitochondrial targeting sequences. Both ATR variants were produced at high levels and purified to apparent homogeneity from soluble cell extracts of recombinant E. coli. The purification consisted of three steps including ammonium sulfate precipitation and ceramic-hydroxyapatite and Mono Q chromatography (Table 3-1). Both ATR variants precipitated between 40 and 50% ammonium sulfate saturation and eluted from the hydroxyapatite and Mono Q columns at 110-135 mM potassium phosphate and 114-130 mM KCl. ATR 239K and 239M were purified 4.5 and 5.4-fold with specific activities of 220 and 190 nmol min-1 mg-1 proteins, respectively. The yields were 14 and 9%. During the purification, inclusion of KCl in chromatography and storage buffers increased enzyme stability. SDS-PAGE was used to follow the purification of both ATR variants. Since purification of both enzymes proceeded similarly, only the gel used to assess the purity of ATR 239K is depicted (Figure 3-2). Following the final Mono Q chromatography step, both variants were homogenous following staining with Coomassie Brilliant Blue.
71 Linearity of the ATR reaction The effect of ATR concentration on activity was determined. Results showed that the rate of adenosylation was proportional to ATR concentration ranging from 5 g to 115 g for both variants and linear regression yielded R2 values of 0.9995 and 0.9993 for ATR 239K and 239M, respectively. ATR Reaction Requirements To determine the ATR reaction requirements, key assay components were individually omitted. For both variants, there was no detectable activity in the absence of ATR, ATP, HOCbl, or titanium (III) citrate (the reducing agent used to produce cob(I)alamin). In the complete reaction mixture, there was 215 and 194 nmol min-1 mg-1 activity for ATR 239K and 239M, respectively. In the absence of MgCl2, activity decreased by 20% for both variants. There was no significant difference in activity in the absence of KCl. Alternative Nucleotide Donors The specificity of the ATR variants for ATP, CTP, GTP, UTP, ADP, and AMP was examined. ATP was found to be the best substrate giving a specific activity of 207 and 197 nmol min-1 mg-1 for ATR 239K and 239M, respectively. These values were set as 100% activity. When CTP, GTP, and UTP were tested as substrates for ATR 239K there was 9, 16, or 8% activity, respectively (Table 3-2). Similar results were found for ATR 239M (6, 14, or 6% activity with CTP, GTP, and UTP, respectively). For both ATR variants there was no detectable activity when AMP or ADP was substituted for ATP. Km and Vmax Values for ATR 239K and 239M Lineweaver-Burk double-reciprocal plots were used to calculate Km and Vmax values (Segel 1976). For ATR 239K and ATR 239M, the Km values for ATP were 6.3 M and
72 6.9 M, and the Km for cob(I)alamin were 1.2 M and 1.6 M, respectively (Figure 3-3). The Vmax values for ATR 239K and 239M were 250 and 200 nmol min-1 mg-1, respectively. When the Km values for cob(I)alamin were determined, saturating levels of ATP (500 M) were added to assay mixtures while varying the concentration of cob(I)alamin. Similarly, saturating levels of cob(I)alamin (50 M) were added to assays when the Km values for ATP were determined. Each value shown in Figure 3-3 is the average of three measurements of the initial reaction rate at the indicated substrate concentrations. MSR Reduces Cob(II)alamin to Cob(I)alamin for AdoCbl Synthesis The human enzyme that catalyzes the third step of CNCbl assimilation, the reduction of cob(II)alamin to cob(I)alamin for AdoCbl synthesis by the ATR enzyme, is unknown. Recently, MSR was shown to catalyze cob(II)alamin reduction for MS activation (Olteanu and Banerjee 2001). To test whether MSR can also reduce cob(II)alamin to cob(I)alamin for AdoCbl synthesis, an in vitro assay was used (MSR-ATR assay). In assays that contained standard components, cob(II)alamin, ATP, DTT, NADPH, 100 g purified MSR, and 10 g purified ATR, AdoCbl was formed at a rate of 0.96 nmol min-1. Controls showed that no measurable AdoCbl was formed in the absence of ATR, MSR, ATP, or cob(II)alamin. When NADPH was excluded from the reaction mixture, 65% of the total activity remained indicating that DTT can partially replace NADPH as the electron donor for this reaction. When DTT was omitted, 100% activity remained with NADPH as the sole electron donor, showing that DTT is not required for the reaction to occur. In addition, several further tests were used to establish that AdoCbl was the product of the reaction. The UV-Visible spectrum of completed
73 reactions was characteristic of AdoCbl, and the reaction product was photolyzed by a 30 min exposure to incandescent light with the formation of cob(II)alamin (Figure 3-4). Moreover, the product of the MSR-ATR reaction co-migrated with authentic AdoCbl by reverse-phase HPLC following co-injection (not shown). These results show that the combination of the MSR and ATR enzymes converted cob(II)alamin to AdoCbl. Since prior studies have shown that the human ATR requires cob(I)alamin for AdoCbl synthesis and is inactive with cob(II)alamin, these results indicate that MSR reduced cob(II)alamin to cob(I)alamin for AdoCbl synthesis by ATR. Cob(I)alamin is Sequestered by the MSR-ATR System Above, we presented evidence that MSR can reduce cob(II)alamin to cob(I)alamin for adenosylation by the ATR. In principle, there are two ways in which this might occur (Figure 3-5). MSR could reduce cob(II)alamin to cob(I)alamin directly, which is subsequently released into solution and diffuses to ATR. Alternatively, MSR could reduce cob(II)alamin bound to ATR and the interaction between the two proteins could sequester cob(I)alamin. To test whether cob(I)alamin was sequestered or released into solution during AdoCbl synthesis by the MSR-ATR system, a chemical trap for cob(I)alamin was used. Iodoacetate (which reacts rapidly and quantitatively with cob(I)alamin to form CMCbl) was added to an MSR-ATR assay at a 1000-fold excess compared to the ATR (400 M compared to 0.4 M). The assay was allowed to proceed to completion. Then, CMCbl and AdoCbl were resolved and quantitated by reverse-phase HPLC. Results showed that 35% of the cob(I)alamin formed was converted to CMCbl by reaction with iodoacetate and that 65% was converted to AdoCbl via the ATR enzyme (Table 3-3, line 1). If "free"
74 cob(I)alamin had been produced under the conditions used, it is expected that the majority should have been converted to CMCbl by reaction with the large excess of iodoacetate prior to diffusion to the ATR for conversion to AdoCbl. Hence, the above results indicated that a significant portion of the cob(I)alamin was sequestered (protected from reaction with iodoacetate) during conversion of cob(II)alamin to AdoCbl by MSR and ATR. As a control, assays were performed that were similar to those described above except that MSR was replaced with the combination of 1 mM DTT plus 50 M FAD to chemically generate cob(I)alamin in situ. Chemical reduction of cob(II)alamin necessarily produces â€œfreeâ€ cob(I)alamin which must then diffuse to the ATR before it can be converted to AdoCbl. Under these conditions, only 10% of the cob(I)alamin formed was converted to AdoCbl while 90% was converted to CMCbl (Table 3-3, line 2). This was in contrast to results obtained when MSR was used to generate cob(I)alamin for the ATR enzyme where 65% of the cob(I)alamin formed was converted to AdoCbl (Table 3-3, line1). Hence these results indicate that the MSR-ATR system sequesters cob(I)alamin during the conversion of cob(II)alamin to AdoCbl. Interestingly, further studies showed that iodoacetate had relatively little effect on the total amount of AdoCbl formed by the MSR-ATR system. In the absence or presence of iodoacetate, 94 or 85 nmol of AdoCbl was produced, respectively (Table 3-3). This indicated that the majority of the cob(I)alamin produced by the MSR-ATR system was sequestered.
75 MSR Produces little Cob(I)alamin in the Absence of the ATR Iodoacetate was also used to test whether MSR can produce â€œfreeâ€ cob(I)alamin in the absence of ATR. Assays were prepared that were similar in composition to the MSR-ATR assay except that ATR was replaced by iodoacetate. During the first 10 min of the reaction, cob(I)alamin was formed at a very low rate (0.02 nmol min-1) (Figure 3-6). Then, at the 10 min time point, 10 g of purified ATR was added to the assay and the rate of cob(I)alamin formation increased approximately 46-fold. These findings showed that the presence of the ATR greatly enhanced the production of cob(I)alamin by MSR suggesting a physical interaction between the MSR and ATR. Control experiments showed that BSA had no effect on the rate of cob(I)alamin formation when substituted for ATR in the second phase of the reaction shown in Figure 3-6. Moreover, the controls showed that iodoacetate was working effectively under our assay conditions. In similar reaction mixtures containing DTT and FAD, the cob(I)alamin formed was efficiently trapped by iodoacetate (data not shown). Stoichiometry of the MSR-ATR system The dependence of ATR activity on the molar ratio of MSR/ATR was determined at ratios from 0 to 40 (Figure 3-7). Maximal activity occurred at a ratio of approximately 4 moles MSR (which is a monomer) per mol ATR monomer. This ratio is similar to the optimal stoichiometry for activation of MS by MSR and is a reasonable value for a physiological process (Olteanu and Banerjee 2001). Ionic Strength Dependence of Cobalamin Reduction and Adenosylation by the MSR-ATR System Variation of the ionic strength from 15 to 720 mM had relatively little effect on the synthesis of AdoCbl by the MSR-ATR system (Figure 3-8). Within this range, greater
76 than 65% activity was retained. This is in contrast to the relatively narrow ionic strength dependence of MS activation by MSR (Olteanu and Banerjee 2001). Discussion Prior studies identified the human ATR and showed that defects in its encoding gene (MMAB) underlie cblB methylmalonic aciduria (Dobson et al. 2002, Leal et al. 2003). Investigations also identified two common polymorphic variants of the ATR that are found in normal individuals (Dobson et al. 2002). Here, both ATR variants were expressed in E. coli, purified, and found to have similar kinetic properties. The specific activities of variants 239K and 239M were 250 and 200 nmol min-1 mg-1, and the Km values were 6.3 and 6.9 M for ATP and 1.2 and 1.6 M for cob(I)alamin, respectively. These values are roughly similar to those previously reported for bacterial ATR enzymes where specific activities range from 53 to 619 nmol min-1 mg-1, and Km values from 2.8 to 110 M for ATP and from 3 to 5.2 M for cob(I)alamin (Suh and Escalante-Semerena 1995, Johnson et al. 2001, Saridakis et al. 2004). The Km values for the human ATR are appropriate to its physiological role since cellular levels of cobalamin are typically 1 M or less in higher organisms and ATP concentrations are in the low mM range (Schneider and Stroinski 1987). Thus, the results reported here show that both common ATR variants are essentially wild-type and have kinetic properties sufficient to meet cellular needs for AdoCbl synthesis. Three classes of ATRs that are unrelated in amino acid sequence have been identified, the PduO-type, CobA-type, and EutT-type (Johnson et al. 2001). Members of the CobA-type and the PduO-type have been purified, partially characterized, and their crystal structures determined (Suh and Escalante-Semerena 1995, Bauer et al. 2001,
77 Johnson et al. 2001, Saridakis et al. 2004). The CobA enzyme has a relatively broad specificity for nucleotide donors. It is active with ATP, CTP, GTP, UTP, and ITP and contains a modified P-loop for nucleotide binding (Bauer et al. 2001). In contrast, the Thermoplasma acidophilum ATR which is a PduO-type enzyme and lacks a recognizable P-loop, uses only ATP and deoxy-ATP as nucleotide donors (Saridakis et al. 2004). Thus, different modes of ATP binding are thought to account for the observed differences in nucleotide specificity by CobAand PduO-type enzymes. The human ATR is a PduO-type enzyme, but results presented here show that its nucleotide specificity is intermediate to that of CobA and T. acidophilum enzymes. In addition, the Km of the human and T. acidophilum enzymes for ATP is 7 versus 110 M suggesting some differences between the two enzymes and the mode of ATP binding which might be expected since these enzymes are only 32% identical in amino acid sequence. Results reported here also showed that an in vitro system containing the human MSR and ATR converted cob(II)alamin to AdoCbl. In this system, MSR reduced cob(II)alamin to cob(I)alamin which in turn was converted to AdoCbl by ATR. When 0.2 M purified ATR and 0.6 M purified MSR was used, the rate of AdoCbl formation was 0.96 nmol min-1. It is probable that this level of activity is sufficient to meet physiological needs. Cells require only very small quantities of AdoCbl. Typical intracellular levels are about 1 M whereas other well-known coenzymes are present at 100 to 1000-fold higher concentrations or more (Schneider and Stroinski 1987). Furthermore, the MSR-ATR system is about 10-fold more active than the analogous bacterial system which consists of the Fpr, FldA, and CobA proteins (Fonseca and Escalante-Semerena 2001). Thus, the MSR-ATR system mediates the conversion of
78 cob(II)alamin to AdoCbl at a physiologically relevant rate. To our knowledge, this is the first reported evidence that MSR can catalyze the reduction of cob(II)alamin to cob(I)alamin for AdoCbl synthesis. In addition to its role in AdoCbl synthesis, prior studies showed that MSR also reduces MS-bound cob(II)alamin to cob(I)alamin for the reductive activation of MS (Leclerc et al. 1998, Olteanu and Banerjee 2001). It is known that MS activation occurs in the cytoplasm but that AdoCbl synthesis occurs in the mitochondrion. Therefore, to carry this dual function MSR must reside in both compartments. Interestingly, recent studies have indicated that alternative transcript splicing leads to the production of MSR enzymes with and without mitochondrial targeting sequences providing further evidence of a dual role of this enzyme (Leclerc et al. 1999). Findings reported here which indicate dual functionality for MSR bring to light an interesting parallel between cobalamin reduction in bacteria and in humans. Biochemical evidence indicated that the E. coli flavodoxin (FldA) catalyzes the reduction of cob(II)alamin to cob(I)alamin for both the reductive activation of MS (MetH) and the synthesis of AdoCbl by the bacterial ATR (CobA) (Fujii and Huennekens 1974, Fujii et al. 1977, Fonseca et al. 2002). Similarly, human MSR reduces cob(II)alamin for both MS activation (Leclerc et al. 1998, Olteanu and Banerjee 2001) and AdoCbl synthesis by the human ATR (this study). Thus, although the human MSR and ATR lack significant sequence similarity to their bacterial counterparts (FldA and CobA), evolution appears to have driven analogous dual physiological roles for both FldA and MSR enzymes. In this report, we also conducted studies to determine whether cob(I)alamin was sequestered or released â€œfreeâ€ in solution during the conversion of cob(II)alamin to
79 AdoCbl by the MSR-ATR system. Experiments in which iodoacetate was used as a chemical trap indicated that cob(I)alamin was sequestered. This is likely to be physiologically important. Cob(I)alamin is one of the strongest nucleophiles that exists in aqueous solution and an extremely strong reductant (E' = -0.61 V) (Schrauzer et al. 1968, Schrauzer and Deutsch 1969, Banerjee et al. 1990). It oxidizes instantaneously in air and rapidly reduces protons to H2 gas at pH 7 (Schneider 1987). Hence, within the cells, sequestration of cob(I)alamin would be important by preventing nonspecific or deleterious side reactions. The finding that cob(I)alamin was sequestered during the conversion of cob(II)alamin to AdoCbl also indicates a physical interaction between the MSR and ATR. This raises the question of whether MSR interacts with both MS and ATR by a conserved mechanism. Prior studies of the MSR-MS reaction indicated that the optimal stoichiometry is ~4 MSR/MS and that activity is maximal over a narrow range of ionic strength indicating that electrostatic interactions are important to the MSR-MS interaction (Olteanu and Banerjee 2001). Here we found that the optimal ratio for the MSR-ATR system is also ~4 MSR/ATR, but in contrast to the MSR-MS system, there was no strict dependence on ionic strength. Hence, although MSR has a conserved function in both systems (cob(II)alamin reductase), the details of the protein-protein interactions between MSR and either MS or ATR are apparently somewhat different. The investigations reported here support prior studies that indicate redundant systems for mitochondrial cobalamin reduction in mammals. Several lines of evidence indicated a role for MSR in the reduction of cob(II)alamin to cob(I)alamin for AdoCbl synthesis: the MSR and ATR enzymes converted cob(II)alamin to AdoCbl at a significant
80 rate; the stoichiometry needed for maximal activity was reasonable (4 MSR/ATR); and cob(I)alamin was sequestered as is expected for the physiologically relevant system. On the other hand, prior investigations showed that patients with inherited defects in MSR (cblE disorder) generally have homocystinuria with the exception of one reported case resulting in combined homocystinuria and mild methylmalonic aciduria (Wilson et al. 1999). This indicates that in vivo cob(II)alamin reduction for AdoCbl synthesis can occur independently of MSR. Of the known complementation groups that result in methylmalonic aciduria, only the cblH and cblA groups have phenotypes consistent with a role in mitochondrial cobalamin reduction. Recent studies have indicated that the cblA group functions to protect MCM from inactivation (Korotkova and Lidstrom 2004) and there has only been one reported case of cblH methylmalonic aciduria (Watkins et al. 2000, Dobson et al. 2002). Thus at this time, there are no strong indications as to the identity of the alternative reductase(s). Redundant systems for cobalamin metabolism are known in bacterial systems (Fonseca and Escalante-Semerena 2000, Johnson et al. 2001), and unpublished results). There, inducible systems provide extra capacity during times of high demand. Similarly, in humans multiple cobalamin reductases might be required for an efficient response to physiological stresses of diets that result in higher rates of propionyl-CoA formation. For example, MSR could be targeted to the mitochondria under circumstances where the diet is rich in amino acids metabolized via propionyl-CoA.
81 CNCblGSCblcob(II)alaminAdoCblcob(I)alamincob(III)alaminreductase ATRbreakdown (AdoCbl is unstable in vivo)Methioninesynthase[cob(II)alamin]Methioninesynthase[CH3Cbl] inactivationMSR -ligandtransferaseCH3THF +homocysteineTHF +methioninepropionyl-CoA(2S)-methylmalonyl-CoA(2R)-methylmalonyl-CoAsuccinyl-CoA MSRMCMPCCMCEE CENTRAL METABOLISM CNCblGSCblcob(II)alaminAdoCblcob(I)alamincob(III)alaminreductase ATRbreakdown (AdoCbl is unstable in vivo)Methioninesynthase[cob(II)alamin]Methioninesynthase[CH3Cbl] inactivationMSR -ligandtransferaseCH3THF +homocysteineTHF +methioninepropionyl-CoA(2S)-methylmalonyl-CoA(2R)-methylmalonyl-CoAsuccinyl-CoA MSRMCMPCCMCEE CENTRAL METABOLISM Figure 3-1. Propionyl-CoA metabolism, methionine synthesis, and intracellular cobalamin metabolism. In humans, the pathways shown are needed for the complete catabolism of compounds degraded via propionyl-CoA and for recycling homocysteine. Abbreviations: PPC, propionyl-CoA carboxylase; MCEE, methylmalonyl-CoA epimerase; MCM, AdoCbl-dependent methylmalonyl-CoA mutase; CNCbl, vitamin B12; GSCbl, glutathionylcobalamin, MSR, methionine synthase reductase; ATR, ATP:cob(I)alamin adenosyltransferase; THF, tetrahydrofolate.
Table 3-1. ATP:cob(I)alamin adenosyltransferase activity during ATR 239K and 239M purification Purification step Total protein (mg) Specific activity (nmol min-1 mg-1) Total activity (nmol min-1) Yield (%) Purification (fold) 239K 239M 239K 239M 239K 239M 239K 239M 239K 239M Cell free extract 160 180 49 35 7840 6300 100 100 1 1 Ammonium sulfate precipitate 60 35 110 70 6600 2450 84 39 2.2 2 Hydroxyapatite 10 9 118 97 1180 873 15 14 2.4 2.8 Mono Q 5 3 220 190 1100 570 14 9 4.5 5.4 82
83 21344531211479766kDa 5 Figure 3-2. Purification of the human ATR. SDS-PAGE was used to assess the purification of ATR 239K. Lane 1: molecular mass markers, lane 2: 10 g of soluble cell free extract from the E. coli expression strain, lane 3: 5 g of ammonium sulfate precipitate, lane 4: 4 g hydroxyapatite eluate, lane 5: 2 g of Mono Q fraction containing purified ATR. The purification of ATR 239M proceeded similarly (not shown).
84 Table 3-2. Use of alternative nucleotide donors by the human ATR Activity % Nucleotide Donora ATR 239K ATR 239M ATP (adenosine-5â€™-triphosphate) 100 100 ADP (adenosine-5â€™-diphosphate) NDb ND AMP (adenosine-5â€™-monophosphate) ND ND CTP (cytidine-5â€™-triphosphate) 9 6 GTP (guanosine-5â€™-triphosphate) 16 4 UTP (uridine-5â€™-triphosphate) 8 6 aATP is the physiological substrate for the ATR and activity with this nucleotide was set to 100%. bND, none detected
85 Table 3-3. The MSR-ATR system sequesters cob(I)alamin Amount detected (nmol) Variable assay componentsa AdoCbl CMCbl HOCbl NADPH, MSR, ATR, and iodoacetate 85 46 45 DTT, FAD, ATR, and iodoacetate 18 165 6 NADPH, MSR, and ATR 94 NDb 81 aReaction mixtures contained 200 mM Tris (pH 8.0) 1.6 mM KPi, 2,8 mM MgCl2, 100 mM KCl, 0.1 mM (200 nmol) cob(II)alamin, 0.4 mM ATP, and the components indicated in the table in the following amounts: 10 g purified ATR, 150 g purified MSR, 1 mM NADPH, 1 mM DTT, 50 M FAD. Cob(I)alamin was generated enzymatically using NADPH and purified MSR (lines 1 and 3) or chemically by the combination of DTT and FAD which necessarily generated â€œfreeâ€ cob(I)alamin (line 2). HOCbl, CMCbl, and AdoCbl were resolv ed and quantified by HPLC. In the reverse-phase system used, their re tention times were 11.9, 13.8, and 15.3 min, respectively. bND, none detected.
86 ATR Activity (nmol min-1mg-1)A.[ATP], (M)1 / Rate1 / [ATP] 0501001502000100200300400500 00.0030.0060.0090.01200.050.10.150.2 [cob(I)alamin], (M)B. 05010015020025001020304050 00.001750.00350.005250.00700.10.20.30.40.5 1 / Rate1 / [cob(I)alamin] 0408012016020001020304050 00.0030.0060.00900.10.20.30.40.5 1 / Rate1 / [cob(I)alamin] 00.00300.00600.00900.012000.030.050.080.100.13 1 / Rate1 / [ATP] 0501001502000100200300400500 C.D.ATR Activity (nmol min-1mg-1)ATR Activity (nmol min-1mg-1)ATR Activity (nmol min-1mg-1)[ATP], (M)[cob(I)alamin], (M)ATR Activity (nmol min-1mg-1)A.[ATP], (M)1 / Rate1 / [ATP] 0501001502000100200300400500 00.0030.0060.0090.01200.050.10.150.2 0501001502000100200300400500 0501001502000100200300400500 00.0030.0060.0090.01200.050.10.150.2 00.0030.0060.0090.01200.050.10.150.2 [cob(I)alamin], (M)B. 05010015020025001020304050 00.001750.00350.005250.00700.10.20.30.40.5 1 / Rate1 / [cob(I)alamin] 05010015020025001020304050 05010015020025001020304050 00.001750.00350.005250.00700.10.20.30.40.5 1 / Rate1 / [cob(I)alamin] 00.001750.00350.005250.00700.10.20.30.40.5 00.001750.00350.005250.00700.10.20.30.40.5 1 / Rate1 / [cob(I)alamin] 0408012016020001020304050 0408012016020001020304050 00.0030.0060.00900.10.20.30.40.5 00.0030.0060.00900.10.20.30.40.5 1 / Rate1 / [cob(I)alamin] 00.00300.00600.00900.012000.030.050.080.100.13 1 / Rate1 / [ATP] 00.00300.00600.00900.012000.030.050.080.100.13 00.00300.00600.00900.012000.030.050.080.100.13 1 / Rate1 / [ATP] 0501001502000100200300400500 0501001502000100200300400500 C.D.ATR Activity (nmol min-1mg-1)ATR Activity (nmol min-1mg-1)ATR Activity (nmol min-1mg-1)[ATP], (M)[cob(I)alamin], (M) Figure 3-3. Determination of kinetic constants for ATR 239K and ATR 239M. Panels A and B are for ATR 239K and panels C and D are for ATR 239M. ATR assays were performed as described in the m aterials and m ethods except that the ATP and cob(I)alamin concentrations were varied as indicated. The insets are Lineweaver-Burk plots of the data shown in the larger graph.
87 0.00.10.20.30.40.5440480520560600 AbsorbanceWavelength, (nm) 400 0.00.10.20.30.40.5440480520560600 AbsorbanceWavelength, (nm) 400 Figure 3-4. Absorbance spectra of an MSR-ATR assay. Prior to the addition of NADPH the spectrum is that of cob(I)alamin (); 1 hour after the addition of NADPH and incubation at 37C, the spectrum is that of AdoCbl (â€”â€”); after photolysis, the spectrum is characteristic of cob(II)alamin ( ).
88 cob(I)alamincob(II)alaminNADPH + H+NADP+ MSRAdoCbl+ PPPiATPATR Scheme 1: Cob(I)alamin is released in solution and diffuses to the ATRScheme 2: MSR reduces ATR-bound cob(II)alaminATR + cob(II)alamin AdoCbl+ PPPi+ MSR+ ATRATRâ€”cob(II)alamintight complex NADPH + H++ATPNADP+ MSRATRtight complexcob(I)alamincob(II)alaminNADPH + H+NADP+ MSRAdoCbl+ PPPiATPATR cob(I)alamincob(II)alaminNADPH + H+NADP+ MSRAdoCbl+ PPPiATPATR Scheme 1: Cob(I)alamin is released in solution and diffuses to the ATRScheme 2: MSR reduces ATR-bound cob(II)alaminATR + cob(II)alamin AdoCbl+ PPPi+ MSR+ ATRATRâ€”cob(II)alamintight complex NADPH + H++ATPNADP+ MSRATRtight complexATR + cob(II)alamin AdoCbl+ PPPi+ MSR+ ATRATRâ€”cob(II)alamintight complex NADPH + H++ATPNADP+ MSRATRtight complex Figure 3-5. Two schemes depicting the conversion of cob(II)alamin to AdoCbl by the MSR-ATR system. In scheme 1, MSR reduces cob(II)alamin to cob(I)alamin and releases it free in solution for diffusion to the ATR. This scheme seems unlikely due to the high nucleophilic nature of cob(I)alamin. In scheme 2, MSR reduces cob(II)alamin while bound to the ATR thereby sequestering the highly reactive cob(I)alamin molecule and protecting it from oxidation and then adenosylation can occur. MSR, a dual flavoprotein, mediates the one electron transfer to cob(II)alamin from the two-electron donor, NADPH.
89 Time, (min)ATR addition 0.200.210.220.230.2405101520Wavelength, (525 nm) Time, (min)ATR addition 0.200.210.220.230.2405101520Wavelength, (525 nm) Figure 3-6. MSR produces little cob(I)alamin in the absence of the ATR enzyme. During the first phase of the reaction little cob(II)alamin was reduced to cob(I)alamin. During this phase, iodoacetate was used to detect cob(I)alamin. Iodoacetate reacts chemically and quantitatively with cob(I)alamin to form CMCbl and this reaction proceeds with an increase in absorbance at 525 nm. At the 10 min time point, 10 g of ATR was added to the assay mixture and the rate of cob(I)alamin production incr eased approximately 46-fold. This large increase in the rate of cob(I)a lamin formation suggested a physical interaction between the MSR and ATR enzymes. The assay conditions used were similar to those for the MSR-AT R assay and controls showed that iodoacetate worked effectively under these conditions.
90 0.00.40.81.21.62.00102030MSR/ATR,(mole/mole)Activity, (nmolmin-1) 40 0.00.40.81.21.62.00102030MSR/ATR,(mole/mole)Activity, (nmolmin-1) 40 0.00.40.81.21.62.00102030MSR/ATR,(mole/mole)Activity, (nmolmin-1) 40 Figure 3-7. Stoichiometry of the MSR-ATR system. The MSR-ATR assay was used for these studies. 10 g (0.4 nmol) purified ATR was used with the molar ratio of MSR indicated. Maximal activity required ~4 MSR/ATR.
91 0.01.02.03.04.00120240360480600720Ionic strength, (mM)Activity, (nmolmin-1) 0.01.02.03.04.00120240360480600720Ionic strength, (mM)Activity, (nmolmin-1) Figure 3-8. Effect of ionic strength on the MSR-ATR system. The MSR-ATR assay containing 40 g purified ATR and 28 g purified MSR was used for these studies. The ionic strength was varied by the addition of KCl.
CHAPTER 4 PDUP IS A COENZYME-A-ACYLATING PROPIONALDEHYDE DEHYDROGENASE ASSOCIATED WITH THE POLYHEDRAL BODIES INVOLVED IN B12-DEPENDENT 1,2-PROPANEDIOL DEGRADATION BY SALMONELLA ENTERICA SEROVAR TYPHIMURIUM LT2 Introduction Virtually all Salmonella degrade 1,2-propanediol in a coenzyme-B12-dependent manner and this ability (which is absent in Escherichia coli) is thought to be an important aspect of the Salmonella-specific lifestyle (Roth et al. 1996, Price-Carter et al. 2001). The degradation of 1,2-propanediol may play a role in the interaction of Salmonella with its host organisms. In Salmonella enterica, in vivo expression technology (IVET) has indicated that 1,2-propanediol utilization (pdu) genes may be important for growth in host tissues, and competitive index studies with mice have shown that pdu mutations confer a virulence defect (Conner et al. 1998, Heithoff et al. 1999). 1,2-Propanediol is likely to be abundant in anoxic environments, such as the large intestine, since it is produced by the anaerobic degradation of the common plant sugars rhamnose and fucose. In addition, 1,2-propanediol degradation by S. enterica provides an important model system for understanding coenzyme-B12-dependent processes some of which are important in human physiology, industry, and the environment (Roth et al. 1996). On the basis of biochemical studies, a pathway for coenzyme-B12-dependent 1,2-propanediol degradation by S. enterica was proposed (Figure 1-6). This pathway begins with the conversion of 1,2-propanediol to propionaldehyde, a reaction that is catalyzed by coenzyme-B12-dependent diol dehydratase (Toraya et al. 1979, Obradors et 92
93 al. 1988). The aldehyde is then disproportionated to propanol and propionic acid by a reaction series thought to involve propanol dehydrogenase, coenzyme-A (CoA)-dependent propionaldehyde dehydrogenase, phosphotransacylase, and propionate kinase (Toraya et al. 1979, Obradors et al. 1988, Palacios et al. 2003). This pathway generates one ATP, an electron sink, and a 3-carbon intermediate (propionyl-CoA), which can feed into central metabolism via the methyl-citrate pathway (Horswill and Escalante-Semerena 1997). In S. enterica, the degradation of 1,2-propanediol occurs aerobically, or anaerobically when tetrathionate is supplied as an electron acceptor (Price-Carter et al. 2001). The genes specifically required for growth of S. enterica on 1,2-propanediol are organized as a single contiguous cluster named the propanediol utilization (pdu) locus. Determination of the DNA sequence of this locus showed that 1,2-propanediol degradation was much more complex than prior biochemical studies had suggested. DNA sequence analyses indicated the pdu locus included 23 genes (Bobik et al. 1997, Bobik et al. 1999): six pdu genes are thought to encode enzymes needed for the 1,2-propanediol degradative pathway (Bobik et al. 1997); two are involved in transport and regulation (Bobik et al. 1992, Chen et al. 1994); two are probably involved in diol dehydratase reactivation (Bobik et al. 1999); one is needed for the conversion of vitamin B12 (CN-B12) to coenzyme B12 (Johnson et al. 2001); five are of unknown function; and seven share similarity to genes involved in the formation of carboxysomes, polyhedral bodies found in certain cyanobacteria and some chemoautotrophs (Shively and English 1991, Shively et al. 1998).
94 The finding that the pdu locus included a number of genes similar to those involved in carboxysome formation suggested that a related polyhedral body might be involved in B12-dependent 1,2-propanediol degradation. Indeed, S. enterica was recently shown to form polyhedral bodies (also called polyhedral organelles) during growth on 1,2-propanediol (Bobik et al. 1999, Havemann et al. 2002). In general appearance, the pdu polyhedra are similar to carboxysomes. They are 100-150 nm in cross-section and are composed of a proteinaceous interior covered by a 3to 4-nm protein shell. However, carboxysomes and the pdu polyhedra differ in several ways. Carboxysomes, which contain most of the cellâ€™s ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), function to improve autotrophic growth at low CO2 concentrations and are thought to do so by concentrating CO2 (Shively et al. 2001, Price et al. 2002). By contrast, the pdu polyhedra of S. enterica do not contain RuBisCO. They consist of a protein shell composed in part of the PduA protein (Havemann et al. 2002) and are associated with B12-dependent diol dehydratase and other unidentified proteins (Bobik et al. 1999). The function of the S. enterica polyhedral bodies is uncertain. Work published to date suggests that they may serve to minimize aldehyde toxicity by sequestration and channeling of propionaldehyde and/or by moderating the rate of aldehyde formation through control of diol dehydratase activity (Chen et al. 1994, Rondon et al. 1995, Stojiljkovic et al. 1995, Bobik et al. 1999, Havemann et al. 2002, Havemann and Bobik 2003). If the pdu polyhedra do indeed function to minimize aldehyde toxicity, it might be expected that aldehyde-degrading enzymes are also associated with these structures. In this report, we show that the pduP gene encodes a polyhedral-body-associated
95 CoA-acylating propionaldehyde dehydrogenase important for 1,2-propanediol degradation. Materials and Methods Bacterial Strains, Media, and Growth Conditions The bacterial strains used in this study are listed in Table 4-1. The rich medium used was LB medium (Difco, Detroit, MI) (Miller 1972). The minimal medium used was no-carbon-E (NCE) medium (Vogel and Bonner 1956, Berkowitz et al. 1968) supplemented with 53 mM propanediol, 1 mM MgSO4, 148 nM CNCbl, and 0.3 mM each valine, isoleucine, leucine, and threonine. In addition, for strains carrying pLAC22 media were also supplemented with 1 mM IPTG, and 100 g Amp/ml. Cultures were incubated at 37C with shaking at 250 rpm as previously described (Leal et al. 2003), and cell growth was determined by measuring the optical density of cultures at 600 nm. General Molecular Methods Agarose gel electrophoresis was done as described previously (Sambrook et al. 1989). Plasmid DNA was purified by the alkaline lysis procedure (Sambrook et al. 1989) or by using Qiagen products (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. Following restriction or PCR amplification, DNA was purified using Qiagen PCR purification or gel extraction kits. Restriction enzymes were used according to standard protocols (Sambrook et al. 1989). DNA fragments were ligated using T4 DNA ligase according to the manufacturer's directions. Electroporation was carried out as previously described (Bobik et al. 1999). General Protein Methods SDS-PAGE was performed using Bio-Rad Redigels and Bio-Rad Mini-Protean II electrophoresis units according to the Manufacturer's instructions. Following gel
96 electrophoresis, proteins were stained with Coomassie Brilliant Blue R-250. The protein concentration of solutions was determined using Bio-Rad protein assay reagent (Bio-Rad) (Bradford 1976). P22 Transduction Transductional crosses were performed as described using P22 HT105/1 int-210 (Davis et al. 1980), a mutant phage that has high transducing ability (Schmieger 1971). For the preparation of P22 transducing lysates from strains having galE mutations, overnight cultures were grown on LB-medium supplemented with 11 mM glucose and 11 mM galactose. Transductants were tested for phage contamination and sensitivity by streaking on green plates against P22 H5. Cloning pduP into pLAC22 PCR was used to amplify pduP from the template pMGS2 (Havemann et al. 2002). The primers used for amplification were 5â€™-GGAATTCGGATCCTATGAATACTTCTG AACTCGAAAC-3â€™ (forward) and 5â€™-GGAATTCAAGCTTCAGTTAGCGAATAGAAA AGCC-3â€™ (reverse). These primers introduced BamHI and HindIII restriction sites that were used for cloning into pLAC22 cut with BglII and HindIII (Warren et al. 2000). Cloning was carried out by ligation of the complementary cohesive ends formed by cutting with BamHI and BglII since pduP contains an internal BglII site. Following ligation, clones were introduced into S. enterica strain TR6579 by electroporation and transformants were selected by plating on LB-agar supplemented with ampicillin (Amp) at 100 g/ml. Pure cultures were prepared from selected transformants, and plasmid DNA isolated from these strains was cut with NruI, PstI, and HindIII. The DNA sequence of one clone that released products of the expected sizes following restriction (2,635, 3,322 and 943 base pairs) was determined and found to be identical to the
97 previously published DNA sequence of pduP (Bobik et al. 1999). This clone (pNL9) as well as pLAC22 without insert were moved into strain BE191 by P22 transduction and the resulting strains (BE270 and BE269) were used for complementation studies. Construction of His8-PduP PCR was used to fuse an N-terminal His-tag to the PduP protein. Template pMGS2 was used with the following primers: 5â€™-GGGGATCCATG(CAT)8ATGAATACTTCTGAACTCGAAAC-3â€™ (forward); and 5â€™-GGAATTCAAGCTTCAGTTAGCGA ATAGAAAAGCC-3â€™ (reverse). These primers introduced BamHI and HindIII restriction sites that were used for cloning into pTA925 cut with BglII and HindIII. The ligation mixture was used to transform E. coli strain DH5 and transformants were selected by plating on LB agar supplemented with 25 g kanamycin/ml. Plasmid DNA isolated from selected transformants was analyzed by restriction mapping and DNA sequencing. One plasmid encoding His8-PduP (pNL69) was introduced into E. coli expression strain BL21 (DE3) RIL by electroporation to form expression strain BE273. Purification of His8-PduP under Denaturing and Nondenaturing Conditions For purification of PduP under denaturing conditions, E. coli strain BE273 was grown in 500 ml LB Kan (25 g/ml) broth incubated at 37C with shaking at 275 rpm in a 1-liter baffled Erlenmeyer flask. Cells were grown to an optical density of 0.6-0.8 at 600 nm and PduP production was induced by addition of IPTG to 1 mM. Cells were incubated for an additional 2 hours, and harvested by centrifugation at 6,690 x g for 10 minutes using a Beckman JLA-10.500 rotor. Two grams of cells (wet weight) were resuspended in 3 ml of 50 mM sodium phosphate, 300 mM NaCl, pH 7 and broken using
98 a French pressure cell at 20,000 psi (SLM Aminco, Urbana, IL). The protease inhibitor phenylmethylsulfonylflouride was added to the cell extract to a concentration of 100 g/ml. To separate soluble and insoluble fractions, cell extract was centrifuged at 31,000 x g for 30 minutes using a Beckman JA-20 rotor. Inclusion bodies were purified from the insoluble fraction using BPER-II according to the manufacturerâ€™s instructions. Purified inclusion bodies were solubilized overnight in binding buffer (10 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl pH 7.9, and 6 M urea) and filtered using a 0.45-m filter. Affinity purification was carried out using 1-ml Amersham Pharmacia Hi-trap chelating (Ni2+) column according the manufacturerâ€™s instructions with the following modifications: 6 M urea was added to all buffers, and two wash steps were used, the first with 20 mM imidazole and the second with 60 mM imidazole. The PduP protein was then eluted using 150 mM imidazole. For purification of PduP under nondenaturing conditions, inclusion bodies (isolated as described above) were resuspended in 50 mM potassium phosphate, pH 7, 25 mM NaCl and stored at 4 C for 24 h. Insoluble proteins were pelleted by centrifugation and PduP that remained in the supernatant was purified by nickel-affinity chromatography using Ni-NTA resin (Qiagen) according to the manufacturer's instructions. Briefly, the column was washed with 5 bed volumes of binding buffer (50 mM sodium phosphate, 300 mM NaCl, pH 7) and PduP was eluted with binding buffer supplemented with 40 mM imidazole. Propionaldehyde Dehydrogenase Assays Two assays for propionaldehyde dehydrogenase activity were used. Assay one was carried out as previously described (Walter et al. 1997). Reaction mixtures contained 50
99 mM CHES (2-[N-cyclohexylamino]ethanesulfonic acid) pH 9.5, 10 mM propionaldehyde, 1 mM dithiothreitol (DTT), 75 M NAD+, 100 M HS-CoA, and an appropriate amount of cell extract. Assays were incubated at 37C. Enzyme activity was measured by following the conversion of NAD+ to NADH by monitoring the absorbance of reaction mixtures at 340 nm, and the amount of NADH formed was determined using 340 = 6.22 mM-1 cm-1. Assay two was done as described above except that DTT was omitted and the absorbance of reaction mixtures was followed at 232 nm. An increase in absorbance at 232 nm occurs when thioester bonds are formed and 232 4.5 mM-1 cm-1 (Dawson et al. 1969). Identification of Propionyl-CoA a Product of the Propionaldehyde Dehydrogenase Reaction Propionaldehyde dehydrogenase reactions were performed as described above except that CHES buffer was replaced by 50 mM potassium phosphate, pH 7. Immediately after the reactions reached completion, they were analyzed by reverse-phase HPLC using conditions previously described (Bobik and Rasche 2003). For the purification of reaction products, HPLC solvents were buffered with 10 mM ammonium formate, pH 6.4. Selected reaction products were collected, lyophilized, resuspended in distilled water at a concentration of 4 M. For the MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry) analyses, samples were mixed 1:1 with 10 mg/ml -cyano-4-hydroxycinnamic acid matrix in 0.1% trifluoroacetic acid in 30% acetonitrile. The sample (1 l) was spotted on a MALDI plate and analyzed using an Applied Biosystems Voyager DE-Pro MALDI-TOF MS operated in reflector mode with a delay of 100 nsec, acc voltage of 20 KV, and a grid voltage of 71.5%. The
100 sample was irradiated with a nitrogen laser (337 nm) and 100 individual laser shots were collected for each sample. Construction of a Nonpolar pduP Deletion Bases 28 to 1,386 of the pduP coding sequence were deleted via a PCR-based method (Miller and Mekalanos 1988). The deletion was designed to leave the predicted translational start and stop signals of all pdu genes intact. The following primers were used for PCR amplification of the flanking regions of the pduP gene: primer 1, 5â€™-GCTCTAGACCAGGCCAACATCATCCGTGAAGTTAG-3â€™; primer 2, 5â€™-TCATCGCGACCTCAGCAGGGTTTCGAGTTCAGAAGTAATCATTG-3â€™; primer 3, 5â€™-CGCTAACTGAGGTCGCGATGAATACC-3â€™; and primer 4, 5â€™-GAAGAGCTCAATTCTGCGGCGGTACGCTGACCACC-3â€™. Primers 1 and 2 were used to amplify 496 DNA bases upstream of the pduP gene, and primers 3 and 4 were used to amplify 508 DNA bases downstream of pduP gene. The upstream and downstream amplification products were purified and then fused by a PCR reaction that included 1 ng/l of each product and primers 1 and 4. The fused product was cut with XbaI and SacI (these sites were designed into primers 1 and 4, respectively), and ligated to suicide vector pCVD442 (Miller and Mekalanos 1988) that had been similarly cut. The ligation mixture was used to transform E. coli S17.1 by electroporation and transformants were selected on LB medium supplemented with Amp (100 g/ml). Plasmids were extracted from four of the isolated transformants, screened by restriction analysis, and all released an insert of the expected size (1004 bp). One of these transformants was used to introduce the pduP deletion into the S. enterica chromosome using the procedure of Miller and Mekelanos (1988) with the following modification.
101 For the conjugation step, strain BE47 was used as the recipient and exconjugants were selected by plating on LB agar supplemented with ampicillin (100 g/ml) and chloramphenicol (20 g/ml). Deletion of the pduP coding sequence was verified by PCR using chromosomal DNA as a template. Lastly, the thr-480 dCAM insertion used for selection of exconjugants was crossed-off by P22 transduction using a phage lysate prepared with the wild-type strain and by selecting for prototrophy on NCE glucose minimal medium. Antibody Preparation His8-PduP purified using Ni2+-affinity chromatography was resolved on a 12% Tris-HCl SDS-PAGE gel. The protein band corresponding to His8-PduP was excised and used as a source of antigen for the preparation of polyclonal antibody in a New Zealand white rabbit by Cocalico Biologicals (Reamstown, PA). To eliminate antibodies cross-reacting with E. coli proteins, the antiserum was preadsorbed using acetone powder prepared from E. coli BL21DE3 RIL containing pTA925 (the expression strain lacking pduP) as described previously (Harlow and Lane 1988). Western Blots Cultures were grown and prepared for SDS-PAGE and electroblotting as previously described (Havemann et al. 2002). Membranes were probed using adsorbed anti-PduP antiserum (diluted 1:3,500 in blocking buffer) as the source of the primary antibody, and goat anti-rabbit conjugated to alkaline phosphatase (diluted 1:3,000 in blocking buffer) as the secondary antibody. Chromogenic developing agents were used following the manufacturerâ€™s instructions (Bio-Rad).
102 Electron Microscopy For electron microscopy, cells were grown in minimal medium supplemented with 37 mM succinate and 26 mM propanediol. Cultures (10 ml) were incubated in 125-ml Erlenmeyer flasks at 37 C, with shaking at 275 rpm in a New Brunswick C24 Incubator Shaker. For immunogold localization of the PduP protein, cells were prepared as previously described (Bobik et al. 1999). The source of the primary antibody was rabbit polyclonal anti-PduP antiserum or preimmune serum diluted in 1:100 in PBS. The secondary antibody used was goat anti-rabbit IgG conjugated to12-nm colloidal gold (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) diluted 1:30 in PBS. DNA Sequencing and Analysis DNA sequencing was carried out at the University of Florida Interdisciplinary Center for Biotechnology Research DNA Sequencing Core Facility using Applied Biosystems automated sequencing equipment (Perkin Elmer, Norwalk, CT) or at the University of Florida, Department of Microbiology and Cell Science DNA Sequencing Facility using a LI-COR model 4000L DNA sequencer, automated sequencing equipment, and Base ImagIR Analysis Software version 04.1h (LI-COR, Lincoln, NE). The template for DNA sequencing was plasmid DNA purified using Qiagen 100 tips or Qiagen mini-prep kits. BLAST software was used for sequence similarity searches (Altschul et al. 1997). Chemicals and Reagents Formaldehyde, (R, S)-1,2-propanediol, and antibiotics were from Sigma Chemical Company (St. Louis, MO). Isopropyl--D-thiogalactopyranoside (IPTG) was from Diagnostic Chemicals Limited (Charlotteville PEI, Canada). Restriction enzymes were
103 from New England Biolabs (Beverly, MA) or Promega (Madison, WI). T4 DNA ligase was from New England Biolabs. Glutaraldehyde was from Tousimis (Rockville, MD), and uranyl acetate was from EM Sciences (Washington, PA). LR White resin was from Ted Pella Inc. (Redding, CA). Acrylamide, agarose, ammonium persulfate, Coomassie Brilliant Blue R-250, EDTA, ethidium bromide, 2-mercaptoethanol, N, N-bis-methylene-acrylamide, powdered milk, SDS, and TEMED were from Bio-Rad (Hercules, CA). Bacterial Protein Extraction Reagent II (BPER-II) was from Pierce (Rockford, IL). Other chemicals were from Fisher Scientific (Pittsburgh, PA). Results Effect of a Precise pduP Deletion on the Growth of S. enterica on Minimal 1,2-Propanediol Medium Prior biochemical studies indicated that a CoA-acylating propionaldehyde dehydrogenase was involved in the degradation of 1,2â€“propanediol by S. enterica (Toraya et al. 1979, Obradors et al. 1988). Recent DNA sequence analyses of the pdu operon (a cluster of genes required for 1,2-propanediol degradation by S. enterica) showed that the PduP protein has sequence similarity to a number of CoA-acylating aldehyde dehydrogenases and is 32% identical in amino acid sequence to the E. coli aldehyde/alcohol dehydrogenase, AdhE (Kessler et al. 1991, Bobik et al. 1999). This suggested that PduP is a CoA-acylating propionaldehyde dehydrogenase needed for the degradation of 1,2-propanediol by S. enterica. To examine the role of PduP in 1,2-propanediol degradation, a precise deletion of the pduP gene was constructed using a PCR-based method, and the effects of this mutation on the growth of S. enterica on 1,2-propanediol/CN-B12 minimal medium were examined. The wild-type strain and strain BE191 (pduP) grew with generation times of
104 about 7.4 and 21.7 hours, respectively, and reached maximum optical densities at 600 nm of about 1.38 and 0.49, respectively. This showed the pduP mutant was significantly impaired for growth on 1,2-propanediol/CNCbl minimal medium. As a control, we tested whether the growth defect of strain BE191 (pduP) could be corrected by the expression of pduP in trans. The generation times of strains BE270 (pduP/pLAC22-pduP) and BE269 (pduP/pLAC22-no insert) on 1,2-propanediol/CNCbl minimal medium were 3.2 and 25.4 hours, respectively, and the maximum optical densities reached by these strains were 1.7 for BE270 and 0.45 for BE269. Hence, expression of pduP in trans corrected the growth defect of pduP mutation on 1,2-propanediol/CNCbl minimal medium. This result indicated that the observed growth defect of BE191 was due to deletion of pduP, but not to polarity or a mutation inadvertently introduced during strain construction. This was the expected result since BE191 was constructed by a PCR-based method designed to produce a precise nonpolar deletion. Strain BE270 was found to grow about 2.2-fold faster than did the wild-type strain. It appeared that pLAC22 enhanced growth of this strain on 1,2-propanediol minimal medium for unknown reasons, since the wild-type strain also grew faster when it carried this vector without insert (not shown). Growth tests were repeated twice with similar results. The fact that BE191 (pduP) was impaired for growth on 1,2-propanediol/CNCbl minimal medium, in conjunction with the control experiments described above indicates that pduP plays an important role in 1,2-propanediol degradation in vivo.
105 Propionaldehyde Dehydrogenase Activity in Wild-type S. enterica and a pduP Mutant To test whether the pduP gene encodes a propionaldehyde dehydrogenase, enzyme assays were performed on cell extracts of wild-type S. enterica and strain BE191 (pduP). The pduP mutant used was the same strain used for the growth studies described above, and assays were conducted on both the supernatants and the pellets that resulted from centrifugation of cell extracts at 31,000 x g (Table 4-2). For the wild-type strain, the majority of the propionaldehyde dehydrogenase activity was found in the 31,000 x g pellet which had a specific activity 1.15 mol min-1 mg-1 protein. Similar extracts from the pduP mutant had only 1% of the specific activity of the wild-type strain (0.011 mol min-1 mg-1). When the substrates, propionaldehyde, NAD+ or HS-CoA were omitted from the assay mixture, no activity was detected. To our knowledge, this was the first experimental evidence that the pduP gene encodes a CoA-acylating propionaldehyde dehydrogenase. It was of interest that the majority of propionaldehyde dehydrogenase activity was found in the 31,000 x g pellet from the wild-type strain (99%). Prior studies showed that polyhedral bodies are involved in AdoCbl-dependent 1,2-propanediol degradation by S. enterica (Bobik et al. 1999, Havemann et al. 2002). Hence, the finding that the PduP aldehyde dehydrogenase was associated with the 31,000 x g pellet suggested that it might be associated with the polyhedral bodies which pellet under similar centrifugation conditions (unpublished results). High-level Production of the PduP Protein E. coli strain BE273 (pET41a-His8-pduP) was constructed to produce high levels of recombinant His8-PduP protein. Protein production by this strain as well as by the
106 control strain BE237 (which is isogenic to BE273 except that it contains the expression plasmid without insert) was analyzed by SDS-PAGE (Figure 4-1). Both the soluble and inclusion-body fractions of cell extracts were examined. High amounts of a protein with a molecular mass near 50 kDa were found in the inclusion-body fraction of cell extracts from expression strain BE273, and moderate amounts of a 50-kDa protein were also found in the soluble fraction of cell extracts from this strain (Figure 4-1, lanes 3 and 5). This is near the predicted molecular mass of His8-PduP (50 kDa). In contrast, the soluble and inclusion-body fractions from control strain BE237 (expression vector without insert) contained relatively little protein of 50 kDa molecular mass (Figure 4-1, lanes 2 and 4) indicating that the observed 50-kDa protein produced by BE273 was His8-PduP. The cell extracts analyzed by SDS-PAGE (Figure 4-1) were tested for propionaldehyde dehydrogenase activity. Substantial activity (2.4 mol min-1 mg-1 protein) was found in inclusion bodies isolated from strain BE273, and in the soluble fraction of this strain (0.19 mol min-1 mg-1 protein). However, little activity was found in either the soluble or inclusion-body fractions of control strain BE237 (0.02 mol min-1 mg-1 protein). For the inclusion-body fraction from the expression strain, propionaldehyde dehydrogenase activity was linear with protein concentration (data not shown). Furthermore, when the substrates, propionaldehyde, NAD+ or HS-CoA were omitted from the assay mixture, no activity was detected. Thus, these data provided additional evidence that PduP is a CoA-acylating propionaldehyde dehydrogenase. Purification of Recombinant His8-PduP Protein It was observed that following storage of purified inclusion bodies at 4 C for 24 hours a significant amount of active His8-PduP protein was eluted into the soluble
107 fraction. Following centrifugation of this preparation at 31,000 x g for 30 min., approximately 0.5 mg of soluble protein remained in the supernatant. Enzyme assays showed that this supernatant contained 9.22 mol min-1 mg-1 propionaldehyde dehydrogenase activity. This sample was further purified by nickel-affinity chromatography. The purity of the resulting preparation was analyzed by SDS-PAGE followed by staining with Coomassie. Results indicated that the His8-PduP obtained was highly purified (Figure 4-1, lane 6) and enzyme assays showed that the preparation was enriched in propionaldehyde dehydrogenase activity, with a specific activity of 15.2 mol min-1 mg-1 protien. The reaction requirements for purified PduP were the same as those described above for the partially purified enzyme. These results demonstrated that the His8-PduP protein had propionaldehyde dehydrogenase activity. Propionyl-CoA is a Product of the PduP Reaction The fact that CoA-SH was required for the propionaldehyde dehydrogenase activity of PduP suggested that propionyl-CoA was a reaction product. Several additional experiments were conducted to test this possibility. Propionaldehyde dehydrogenase reactions containing purified PduP protein were allowed to proceed to completion. Then, reverse-phase HPLC was used to identify CoA-SH and CoA-derivatives present in reaction mixtures. A single CoA compound with a retention time of 10.2 min was detected. Further analyses showed that this compound co-eluted with authentic propionyl-CoA following co-injection and resolution via C18 reverse-phase HPLC. This indicated that propionyl-CoA was a product of the PduP aldehyde dehydrogenase reaction. Next, the reaction product identified as propionyl-CoA by reverse-phase HPLC was analyzed by MALDI-TOF MS. Three major peaks at m/z 824.8, 848.1, and 878.1
108 were observed in the mass spectrum. These peaks correspond to the [M+H]+, [M+H+Na]+, and [M+3NH4]+ of propionyl-CoA, further supporting the assignment of propionyl-CoA as a PduP reaction product. In addition, the propionaldehyde dehydrogenase reaction catalyzed by the PduP enzyme was followed spectrophotometrically at 232 nm, the wavelength at which thioester bonds characteristically absorb (Dawson et al. 1969). Within experimental error, the specific activity of purified PduP was the same when reactions were followed at 340 nm (NAD+ reduction) or 232 nm (thioester bond formation). Thus, based on the evidence described above, we conclude that propionyl-CoA is a product of the PduP propionaldehyde dehydrogenase reaction. Preparation and Specificity of the Anti-PduP Antiserum To obtain the antigen needed for preparation of antisera, His8-PduP was purified from inclusion bodies isolated from expression strain BE273 by Ni2+-affinity chromatography under denaturing conditions. SDS-PAGE indicated that the PduP protein obtained was highly purified (data not shown). Denaturing conditions were used to obtain antigen because a nondenaturing protocol was unavailable at the time. Following purification, His8-PduP was used to prepare polyclonal anti-PduP antiserum in rabbit. The antiserum obtained was subjected to a preadsorption procedure to remove cross-reacting proteins (Warren et al. 2000). To determine the specificity of the adsorbed anti-PduP antiserum, Western blot analysis was done on boiled cell lysates. The anti-PduP antibody preparation recognized one major protein band in extracts from wild-type S. enterica at approximately 50 kDa (Figure 4-2, lane 2); however, this band was not detected in strain BE191, which contained a nonpolar pduP deletion, but was otherwise isogenic to the wild-type strain (Figure 4-2, lane 2). This indicated that the
109 antiserum obtained was specific for the PduP protein. Furthermore, the anti-PduP antibody preparation detected a 50-kDa band in boiled cell extracts from PduP expression strain BE270 (pLAC22-PduP) but not in extracts from strain BE269 which carried the pLAC22 expression plasmid without the insert but is otherwise isogenic to strain BE270 (Figure 4-2, lanes 4 and 5). This provided additional evidence that the antibody preparation was specific for the PduP protein. The minor band observed in Figure 4-2, lane 5, was most likely a degradation product of PduP that resulted from overproduction since this band was not detected in extracts from the wild-type strain (Figure 4-2, lane 2). Localization of PduP Immunoelectron Microscopy Wild-type S. enterica and BE191 (pduP) were grown on succinate/1,2-propanediol minimal medium which induces formation of the polyhedral bodies involved in 1,2-propanediol degradation (Bobik et al. 1999, Havemann et al. 2002). Immunogold labeling of wild-type S. enterica and BE191 (pduP) was then carried out using anti-PduP antiserum described above. In the micrograph (Figure 4-3), the antibody-conjugated gold particles (solid black circles) indicate the location of the PduP protein. Note that the gold particles localized to the polyhedral bodies (which appear as uniformly stained regions within the cytoplasm) in the wild-type strain, but no labeling was observed in the pduP mutant, although polyhedra were present. These results indicated the antibody preparation reacted specifically with the PduP protein under the labeling conditions used and that the PduP protein localizes to the polyhedral bodies. Additional electron microscopy studies of standard thin sections showed that the pduP mutant formed normal-appearing polyhedra (standard thin sections result in higher contrast than does fixation for immunolabeling and the fine structure of the
110 polyhedra is more easily discerned). This result indicated that PduP is nonessential for polyhedral body formation. Propionaldehyde Dehydrogenase Activity is associated with Purified pdu Bodies To further investigate the subcellular localization of the PduP enzyme, the polyhedral bodies involved in 1,2-propanediol degradation were purified and propionaldehyde dehydrogenase activity was followed during the course of their purification (Table 4-3). The polyhedra were purified by a combination of detergent treatment and differential and density-gradient centrifugation as described in Havemann and Bobik (2003). Electron microscopy and SDS-PAGE indicated that the polyhedra obtained were highly purified (not shown). During the purification the specific activity of the PduP propionaldehyde dehydrogenase increased 13.5-fold from 0.3 4.0 mol min-1 mg-1 protein (Table 4-3). This suggested that the polyhedral bodies comprised about 7% of the total cell protein, which is consistent with previous electron microscopy (Havemann et al. 2002). Furthermore, Western blots verified that the PduP enzyme was associated with the purified polyhedra (not shown). Thus, enzyme assays and Western blots with purified polyhedra supported the immunolabeling studies described above and indicated that the PduP propionaldehyde dehydrogenase is associated with the polyhedral bodies involved in 1,2-propanediol degradation. Discussion Prior biochemical studies indicated that a CoA-acylating propionaldehyde dehydrogenase was required for AdoCbl-dependent 1,2-propanediol degradation (Toraya et al. 1979, Obradors et al. 1988). Subsequent DNA sequence analyses of the pdu operon identified a presumptive enzyme (PduP) that was 32% identical to the E. coli aldehyde/alcohol dehydrogenase, AdhE (Bobik et al. 1999). In this report, genetic and
111 biochemical evidence was presented that established PduP as a CoA-acylating propionaldehyde dehydrogenase involved in AdoCbl-dependent 1,2-propanediol degradation by S. enterica. Previously, Jorge Escalante's group proposed that two propionaldehyde dehydrogenases might be involved in 1,2-propanediol degradation by S. enterica: one that produces propionyl-CoA and a second that oxidizes propionaldehyde directly to propionate (Palacios et al. 2003). The PduP enzyme studied here produced propionyl-CoA as a reaction product and was inactive in the absence of HS-CoA showing that it is incapable of oxidizing propionaldehyde directly to propionate under the assay conditions used. It was also shown that an S. enterica strain with a nonpolar pduP null mutation grew at one-third the rate of the wild-type strain. The residual growth of a pduP mutant could have resulted from the activity of an alternative propionaldehyde dehydrogenase, such as the one proposed to convert propionaldehyde directly to propionate (Palacios et al. 2003), since the propionate produced by this enzyme could be used as a carbon and energy source via the methyl citrate pathway (Horswill and Escalante-Semerena 1997). Previously, we established that S. enterica formed polyhedral bodies during AdoCbl-dependent growth on 1,2-propanediol (Bobik et al. 1999). Immunolabeling studies demonstrated that these bodies consisted of a protein shell (partly composed of the PduA protein) AdoCbl-dependent diol dehydratase and additional unidentified proteins (Bobik et al. 1999, Havemann et al. 2002). In this report, we presented several lines of evidence that indicated PduP is polyhedral-body-associated. The finding that the pdu polyhedra include both diol dehydratase (aldehyde-producing) and propionaldehyde
112 dehydrogenase (aldehyde-consuming) is consistent with the prior proposal that these structures function to minimize aldehyde toxicity (Chen et al. 1994, Stojiljkovic et al. 1995, Havemann et al. 2002). Presumably, the shell of the polyhedral body could trap the propionaldehyde produced by the PduCDE diol dehydratase until it is consumed by the PduP propionaldehyde dehydrogenase. Sequestering propionaldehyde until it is converted to propionyl-CoA might protect sensitive cytoplasmic components. Alternatively, the inclusion of diol dehydratase and propionaldehyde dehydrogenase in the pdu polyhedra might simply serve to juxtapose these enzymes in order to facilitate propionaldehyde channeling, although it is unclear why such a complex structure would be needed for this sole purpose. Prior studies suggested that the pdu polyhedra minimize aldehyde toxicity by limiting aldehyde production through control of AdoCbl availability (Havemann et al. 2002). Such a mechanism would be improved by either aldehyde channeling or sequestration, and a combined mechanism that fine tunes propionaldehyde production and consumption could explain the selective advantage provided by these complex polyhedral bodies involved in 1,2-propanediol degradation.
113 Table 4-1. Bacterial strains Species Strain Genotype E. coli BL21 (DE3) RIL (E. coli B) F-ompT hsdS (rB-mB-) dcm+ Tetr gal (DE3) endA Hte (argU ileY leuW Camr) BE237 BL21 (DE3) RIL/pET-41a (T7 expression vector without insert, Kanr) BE273 BL21 (DE3) RIL/pNL69 (T7expression vector with insert encoding His8-PduP) S17.1pir recA(RP4-2-Tc::Mu)pir BE47 thr-480::Tn10dCam S. enterica serovar Typhimurium LT2 TR6579 metA22 metE551 trpD2 ilv-452 hsdLt6 hsdSA29 HsdBstrA120 GalE-Leu-ProBE191 pduP659 BE268 LT2/ pNL9 (pLAC22-pduP, Apr) BE269 pduP659/ pLAC22 (vector without insert, Apr) BE270 pduP659/pNL9 (pLAC22-pduP, Apr)
114 Table 4-2. Propionaldehyde dehydrogenase activity in extracts from wild-type S. enterica and strain BE191 (pduP). Propionaldehyde dehydrogenase activity Extract S/Pa Total protein (mg) Specific activity (mol min-1 mg-1 protein) Total activity (mol min-1) Total activity (%) S 45 0.004 0.18 1 Wild-type P 25 1.15 29.23 99 S 51 NDb ND 0 BE191 (pduP) P 30 0.011 0.33 100 aAssays were performed on the supernatant (S) and pellet (P) fractions following centrifugation at 31,000 x g. bND indicates, none detected.
115 Table 4-3. Propionaldehyde dehydrogenase activity during polyhedral body purification Sample Protein (mg) Total activity (mol min-1) Specific activitya (mol min-1 mg-1 protein) Yield (%) Fold -purification Crude extract 515 152 0.30 100 1.0 Detergent/salts treatment 338 205 0.61 135 2.1 12,000 x g super 338 169 0.50 111 1.7 48,000 x g pellet 5.0 9.6 1.92 6.3 6.5 12,000 x g super 4.3 8.6 2.00 5.7 6.8 Sucrose density gradient 0.3 1.2 4.00 0.8 13.6 aActivities were calculated from the initial rate
116 22664531971412345kDa 67PduP Figure 4-1. SDS-PAGE analysis of His8-PduP. Lane 1: Molecular mass markers, lane 2: 12 g soluble extract from the control strain (BE237), lane 3: 12 g soluble extract from the His8-PduP expression strain (BE273), lane 4: 12 g inclusion-body extract from the control strain, lane 5: 5 g inclusion-body extract from the His8-PduP expression strain, lane 6: 2 g His8-PduP purified by nickel-affinity chromatography. The control and the expression strains were isogenic except that the control strain lacked the pduP coding sequence.
117 1234kDa 209804935295 Figure 4-2. Western blot using the anti-PduP antiserum. Lane 1: Molecular mass standards, lane 2: wild-type (Salmonella enterica), lane 3: pduP (strain BE191), lane 4: pduP/pLAC22 (vector without insert), lane 5: pduP/pLAC22-pduP. Each lane was loaded with 20 l boiled whole cells (OD600 = 1.0).
118 B B B Figure 4-3. Immunogold localization of the PduP enzyme. A, Wild-type S. enterica; B, strain BE191 (pduP). The 12 nm gold particles (small black circles) indicate the location of the PduP protein. The arrows point to the polyhedral bodies. Bars 100 nm
CHAPTER 5 CONCLUSIONS In humans, cobalamin cofactors are required coenzymes for two enzymatic reactions that are vital to human health. These coenzymes are used in propionate metabolism and methionine biosynthesis, and in humans, deficiencies in cobalamin metabolism result in methylmalonic aciduria and homocystinuria. Nine complementation groups associated with such deficiencies have been identified. Historically, progress on understanding these diseases has been slow due to difficulties in purifying the enzymes involved as well as the lack of facile genetic methods. A bacterial model system was used to circumvent some of these problems and allowed progress on the identification and characterization of the genes and enzymes involved in these rare, but devastating disorders. Identification of the Bovine and Human Adenosyltransferase In Chapter 2, S. enterica was used as a model system for the identification of the bovine and human ATR cDNAs by complementation analysis. In this screening technique, the bovine cDNA was isolated by complementing an ATR deficient S. enterica strain. Subsequently, sequence similarity searches using the bovine ATR cDNA identified a homologous human gene. Both human and bovine expression libraries were screened; however, complementing clones were only found from the bovine library. This could have been due to the relative abundance of ATR mRNA in these systems. Ruminant livers have the highest reported concentration of B12-dependent 119
120 enzymes measured in any tissue. Similar methods could be applied for the identification of additional cDNAs involved in cobalamin metabolism. To determine whether the bovine and human cDNAs that we identified encode ATRs, the cDNAs were cloned and over expressed in E. coli. Cell extracts from the expression strains were then used for biochemical studies. Both the soluble and insoluble fractions of the crude cell extracts of bovine and human expression strains were found to have ATR activity. This suggests that the cDNAs we identified encode ATR enzymes. Additional studies were used to show that the human cDNA could function as an ATR in vivo. An S. enterica ATR mutant transformed with a plasmid-encoded source of the human ATR was used for growth studies on 1,2-propanediol supplemented with HOCbl. From these experiments, we showed that the human ATR restored wild-type phenotype to an ATR-deficient S. enterica strain proving that the human ATR can function physiologically in a heterologous host. Previous studies have shown that patients with cblB methylmalonic aciduria lack ATR activity (Fenton and Rosenberg 1981). To determine if mutations in the ATR underlie cblB methylmalonic aciduria, expression of this enzyme was monitored in normal and cblB mutant fibroblast cells. Western blot analysis using antibodies specific for the ATR showed that expression of this protein was altered in fibroblast cells from patients with cblB when compared to control cells. Additional studies conducted concurrently in another lab showed that the ATR gene (MMAB) corresponded to the cblB complementation group of methylmalonic aciduria (Dobson et al. 2002). Biochemical Characterization of the Human Adenosyltransferase In Chapter 3, we investigated the biochemical properties of the human ATR. Dobson et al. previously analyzed the MMAB gene and identified two amino acid
121 substitutions that are common polymorphic variants in control cell lines (Dobson et al. 2002). Both of these variants were independently expressed, purified, and used for biochemical analysis. In the studies conducted, the variants behaved similarly suggesting that both are normal forms of the enzyme. The kinetic properties including Vmax, and Km for ATP and cob(I)alamin were determined. The Vmax falls within the range of previously reported ATRs including CobA, PduO, and T. acidophilum ATR (Suh and Escalante-Semerena 1995, Johnson et al. 2001, Saridakis et al. 2004). The Km for cob(I)alamin (1 M) is physiologically significant when compared to the intracellular concentration of B12 in human tissue (Schneider and Stroinski 1987). The Km for ATP is low when compared to the physiological levels of this substrate but is considered relevant since ATP is required for many different reactions in human tissue. In this study, the nucleotide specificity of the human ATR variants was also analyzed. It was found that ATR variants are highly specific for ATP (the physiological substrate for adenosylation). This result is not surprising since catalysis with other nucleotide substrates (GTP, CTP, and UTP) resulting in GCbl, CCbl, and UCbl are generally inhibitory to AdoCbl-dependent enzymes and would be counter-productive (Toraya and Mori 1999). Establishing the biochemical properties of control ATRs provides a basis of comparison for the characterization of disease causing variants of this enzyme. Methionine Synthase Reductase is a Cob(II)alamin Reductase for the Human Adenosyltransferase In addition to analyzing the biochemical properties of the human ATR, we also investigated whether human MSR can act as a cob(II)alamin reductase for the synthesis of AdoCbl by the ATR (Chapter 3). Previous studies have shown that MSR functions as
122 a cob(II)alamin reductase for reductive activation of MS (Olteanu and Banerjee 2001). Here, an MSR-ATR linked assay was developed and this provided biochemical evidence that MSR can also function as a cob(II)alamin reductase for AdoCbl synthesis. The rate of AdoCbl formation in these assays (0.96 nmol min-1) is physiologically significant since humans require only small amounts of cobalamin (Schneider 1987). This rate is also significant when compared to previous measurements of AdoCbl formed in human fibroblast cells (0.021 pmol min-1 mg-1) (Fenton and Rosenberg 1981). In mammalian systems, MSR is a cytosolic protein while ATR is localized to the mitochondria. In these studies we demonstrated that MSR is as a cob(II)alamin reductase for the ATR in vitro. The proposed requirement of MSR for AdoCbl synthesis necessitates the presence of MSR in both the cytoplasm and the mitochondria. Recent studies have shown that alternative transcript splicing of MSR produces protein with and without a mitochondrial targeting sequence (Leclerc et al. 1999). These findings strengthen the hypothesis that MSR has dual functionality in the cytoplasm and the mitochondria. Investigations also suggest an interaction between MSR and ATR. In MSR-ATR linked assays containing iodoacetate (an alkylating agent that forms CMCbl in the presence of cob(I)alamin), very little CMCbl was formed suggesting that MSR does not release cob(I)alamin free in solution. In addition, HPLC analyses showed that AdoCbl formation was favored even when a large excess of iodoacetate was added to the assays. These studies indicate that an interaction between MSR and ATR triggers cobalamin reduction.
123 Studies also indicated that humans have redundant systems for mitochondrial cob(II)alamin reduction. Prior complementation analysis demonstrated that cblE (MSR) disorders usually result in homocystinuria without methylmalonic aciduria with only one reported case having both homocystinuria and mild methylmalonic aciduria (Wilson et al. 1999). The fact that most cblE patients lack methylmalonic aciduria suggests that there are redundant mitochondrial cob(II)alamin reductase(s) involved in AdoCbl synthesis. There is precedence for redundant cobalamin reductases in bacteria. The role of MSR could be to provide extra capacity under certain conditions, such as a high protein diet, rich in amino acids that are metabolized via the propionyl-CoA pathway. Future Experimentation The development of the screening method employed in this study can be used in the future as a general technique for the isolation of other cDNAs involved in cobalamin metabolism. Salmonella is an excellent model system for these studies since AdoCbl-dependent diol dehydratase for 1,2-propanediol degradation has been well studied. Once other enzymes involved in cobalamin metabolism are identified in S. enterica, strains deficient in these activities can be used to screen mammalian expression libraries by complementation. Hence, this technique may allow for the identification of other genes crucial for cobalamin metabolism in eukaryotic systems. Now that a purification method has been developed for the ATR, efforts can be focused on crystallizing this protein. Determining the three dimensional structure of the ATR will allow us to understand how mutations in this enzyme lead to dysfunction by comparing amino acid residues in normal and mutant ATRs and mapping the altered amino acid residues onto the structure. Potentially, this process would help lend insight in identifying the residues of the ATR that are crucial for enzymatic activity.
124 Future studies should also be focused on determining the mode of interaction between MSR and ATR. Approaches to verifying the interaction between these enzymes include gel filtration, immuno-precipitation, and native PAGE analyses. All of these techniques would verify a physical interaction between MSR and ATR and give a better understanding of how this system functions in vivo. Most importantly, knowledge of the ATR gene will allow for DNA based methods of methylmalonic aciduria diagnosis following amniocentesis or chorionic villi sampling and could aid in determining the mode of treatment. Additionally, identification of the ATR gene makes possible the development of gene therapy as a treatment for cblB methylmalonic aciduria.
LIST OF REFERENCES Aberg, A., S. Hahne, M. Karlsson, A. Larsson, M. Ormo, A. Ahgren and B. M. Sjoberg. 1989. Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli. J. Biol. Chem. 264:12249-52. Allen, S. G. H., R. W. Kellermeyer, R. L. Stjernholm and H. G. Wood. 1964. Purification and properties of enzymes involved in the propionic acid fermentation. J. Bacteriol. 87:171-187. Altschul, S. F., W. Gish, W. Miller, E. W. Myers and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. Altschul, S. F., T. L. Madden, A. A. Schffer, J. Zhang, Z. Zhang, W. Miller and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. Ampola, M. G., M. J. Mahoney, E. Nakamura and K. Tanaka. 1975. Prenatal therapy of a patient with vitamin B12 responsive methylmalonic acidemia. N. Engl. J. Med. 293:313-317. Baker, S. J. and V. I. Mathan. 1981. Evidence regarding the minimal daily requirement of dietary vitamin B12. Am. J. Clin. Nutr. 34:2423-33. Banerjee, R. 1997. The Yin-Yang of cobalamin biochemistry. Chem. Biol. 4:175-86. Banerjee, R. (eds.). 1999. Chemistry and Biochemistry of B12. John Wiley & Sons, Inc, New York. Banerjee, R. and S. Chowdhury. 1999. Methylmalonyl-CoA mutase, p. 707-730. In R. Banerjee (ed.), Chemistry and Biochemistry of B12. John Wiley & Sons, Inc., New York. Banerjee, R. and S. W. Ragsdale. 2003. The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes. Annu. Rev. Biochem. 72:209-47. Banerjee, R. V., S. R. Harder, S. W. Ragsdale and R. G. Matthews. 1990. Mechanism of reductive activation of cobalamin-dependent methionine synthase: an electron paramagnetic resonance spectroelectrochemical study. Biochemistry 29:1129-35. 125
126 Banerjee, R. V., N. L. Johnston, J. K. Sobeski, P. Datta and R. G. Matthews. 1989. Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain. J. Biol. Chem. 264:13888-95. Banerjee, R. V. and R. G. Matthews. 1990. Cobalamin-dependent methionine synthase. Faseb. J. 4:1450-9. Barker, H. A., H. Weisbach and R. D. Smyth. 1958. A Coenzyme containing pseudovitamin B12. Proc. Natl. Acad. Sci. U S A 44:1039-1097. Battersby, A. R. 1994. How nature builds the pigments of life: the conquest of vitamin B12. Science 264:1551-7. Bauer, C. B., M. V. Fonseca, H. M. Holden, J. B. Thoden, T. B. Thompson, J. C. Escalante-Semerena and I. Rayment. 2001. Three-dimensional structure of ATP:corrinoid adenosyltransferase from Salmonella typhimurium in its free state, complexed with MgATP, or complexed with hydroxycobalamin and MgATP. Biochemistry 40:361-74. Berkowitz, D., J. Hushon, H. Whitfield, Jr., J. Roth and B. Ames. 1968. Procedure for identifying nonsense mutations. J. Bacteriol. 96:215-220. Bobik, T. A., M. Ailion and J. R. Roth. 1992. A single regulatory gene integrates control of vitamin B12 synthesis and propanediol degradation. J. Bacteriol. 174:2253-66. Bobik, T. A., G. D. Havemann, R. J. Busch, D. S. Williams and H. C. Aldrich. 1999. The propanediol utilization (pdu) operon of Salmonella enterica serovar Typhimurium LT2 includes genes necessary for the formation of polyhedral organelles involved in coenzyme B12-dependent 1,2-propanediol degradation. J. Bacteriol. 181:5967-5975. Bobik, T. A. and M. E. Rasche. 2003. HPLC assay for methylmalonyl-CoA epimerase. Anal. Bioanal. Chem. 375:344-9. Bobik, T. A. and R. S. Wolfe. 1989. Activation of formylmethanofuran synthesis in cell extracts of Methanobacterium thermoautotrophicum. J. Bacteriol. 171:1423-1427. Bobik, T. A., Y. Xu, R. M. Jeter, K. E. Otto and J. R. Roth. 1997. Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase. J. Bacteriol. 179:6633-9. Booker, S., S. Licht, J. Broderick and J. Stubbe. 1994. Coenzyme B12-dependent ribonucleotide reductase: evidence for the participation of five cysteine residues in ribonucleotide reduction. Biochemistry 33:12676-85.
127 Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. Brady, R. O., E. G. Castanera and H. A. Barker. 1962. The enzymatic synthesis of cobamide coenzymes. J. Biol. Chem. 237:2325-32. Cauthen, S. E., M. A. Foster and D. D. Woods. 1966. Methionine synthesis by extracts of Salmonella typhimurium. Biochem. J. 98:630-635. Chen, P., D. I. Andersson and J. R. Roth. 1994. The control region of the pdu/cob regulon in Salmonella typhimurium. J. Bacteriol. 176:5474-82. Ciani, F., M. A. Donati, G. Tulli, G. M. Poggi, E. Pasquini, D. S. Rosenblatt and E. Zammarchi. 2000. Lethal late onset cblB methylmalonic aciduria. Crit. Care Med. 28:2119-21. Claros, M. G. and P. Vincens. 1996. Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur. J. Biochem. 241:770-786. Conner, C. P., D. M. Heithoff, S. M. Julio, R. L. Sinsheimer and M. J. Mahan. 1998. Differential patterns of acquired virulence genes distinguish Salmonella strains. Proc. Natl. Acad. Sci. U S A 95:4641-5. Cooper, B. A. and D. S. Rosenblatt. 1987. Inherited defects of vitamin B12 metabolism. Annu. Rev. Nutr. 7:291-320. Crouzet, J., S. Levy-Schil, B. Cameron, L. Cauchois, S. Rigault, M. C. Rouyez, F. Blanche, L. Debussche and D. Thibaut. 1991. Nucleotide sequence and genetic analysis of a 13.1-kilobase-pair Pseudomonas denitrificans DNA fragment containing five cob genes and identification of structural genes encoding Cob(I)alamin adenosyltransferase, cobyric acid synthase, and bifunctional cobinamide kinase-cobinamide phosphate guanylyltransferase. J. Bacteriol. 173:6074-87. Davis, R. W., D. Botstein and J. R. Roth. 1980. Advanced bacterial genetics : a manual for genetic engineering. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Dawson, R. M. C., D. C. Elliott, W. H. Elliott and K. M. Jones (eds.). 1969. Data for biochemical research, 2nd ed. Oxford University Press, Oxford. Debussche, L., M. Couder, D. Thibaut, B. Cameron, J. Crouzet and F. Blanche. 1991. Purification and partial characterization of cob(I)alamin adenosyltransferase from Pseudomonas denitrificans. J. Bacteriol. 173:6300-6302.
128 Del Corral, A. and R. Carmel. 1990. Transfer of cobalamin from the cobalamin-binding protein of egg yolk to R binder of human saliva and gastric juice. Gastroenterologist 98:1460-6. Dixon, M. M., S. Huang, R. G. Matthews and M. Ludwig. 1996. The structure of the C-terminal domain of methionine synthase: presenting S-adenosylmethionine for reductive methylation of B12. Structure 4:1263-75. Dobson, C. M., T. Wai, D. Leclerc, H. Kadir, M. Narang, J. P. Lerner-Ellis, T. J. Hudson, D. S. Rosenblatt and R. A. Gravel. 2002. Identification of the gene responsible for the cblB complementation group of vitamin B12-dependent methylmalonic aciduria. Hum. Mol. Genet. 11:3361-9. Dobson, C. M., T. Wai, D. Leclerc, A. Wilson, X. Wu, C. Dore, T. Hudson, D. S. Rosenblatt and R. A. Gravel. 2002. Identification of the gene responsible for the cblA complementation group of vitamin B12-responsive methylmalonic acidemia based on analysis of prokaryotic gene arrangements. Proc. Natl. Acad. Sci. U S A 99:15554-9. Dolphin, D. 1982. B12. John Wiley & Sons, Inc., New York. Drennan, C. L., S. Huang, J. T. Drummond, R. G. Matthews and M. L. Ludwig. 1994. How a protein binds B12: a 3.0 x-ray structure of B12-binding domains of methionine synthase. Science 266:1669-1674. Drummond, J. T. and R. G. Matthews. 1993. Cobalamin-dependent and cobalamin-independent methionine synthases in Escherichia coli: two solutions to the same chemical problem. Adv. Exp. Med. Biol. 338:687-692. Drummond, J. T. and R. G. Matthews. 1994. Nitrous oxide inactivation of cobalamin-dependent methionine synthase from Escherichia coli: characterization of the damage to the enzyme and prosthetic group. Biochemistry 33:3742-50. Fenton, W. A., R. A. Gravel and D. Rosenblatt. 2000. Disorders of propionate and methylmalonate metabolism. In C. R. Scriver, A. L. Beaudet, W. S. Sly, et al. (ed.), The Metabolic and molecular basis of inherited disease. McGraw-Hill, New York. Fenton, W. A. and L. E. Rosenberg. 1978a. Genetic and biochemical analysis of human cobalamin mutants in cell culture. Annu. Rev. Genet. 12:223-48. Fenton, W. A. and L. E. Rosenberg. 1978b. Mitochondrial metabolism of hydroxocobalamin: synthesis of adenosylcobalamin by intact rat liver mitochondria. Arch. Biochem. Biophys. 189:441-7. Fenton, W. A. and L. E. Rosenberg. 1981. The defect in the cblB class of human methylmalonic acidemia: deficiency of cob(I)alamin adenosyltransferase activity in extracts of cultured fibroblasts. Biochem. Biophys. Res. Commun. 98:283-289.
129 Fenton, W. A. and L. E. Rosenberg. 2000. Inherited Disorders of Cobalamin Transport and Metabolism. In C. R. Scriver, A. L. Beaudet, W. S. Sly, et al. (ed.), The Metabolic and molecular basis of inherited disease. McGraw-Hill, New York. Fonseca, M. V., N. R. Buan, A. R. Horswill, I. Rayment and J. C. Escalante-Semerena. 2002. The ATP : Co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica requires the 2 '-OH group of ATP for function and yields inorganic triphosphate as its reaction byproduct. J. Biol. Chem. 277:33127-33131. Fonseca, M. V. and J. C. Escalante-Semerena. 2000. Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica serovar Typhimurium LT2. J. Bacteriol. 182:4304-9. Fonseca, M. V. and J. C. Escalante-Semerena. 2001. An in vitro reducing system for the enzymic conversion of cobalamin to adenosylcobalamin. J. Biol. Chem. 276:32101-8. Frey, P. A. and G. H. Reed. 2000. Radical mechanisms in adenosylmethionineand adenosylcobalamin-dependent enzymatic reactions. Arch. Biochem. Biophys. 382:6-14. Friedmann, H. C. 1975. Biosynthesis of corrinoids, p. 75-103. In B. M. Babior (ed.), Cobalamin. John Wiley and Sons, New York. Fujii, K., J. H. Galivan and F. M. Huennekens. 1977. Activation of methionine synthase: further characterization of flavoprotein system. Arch. Biochem. Biophys. 178:662-70. Fujii, K. and F. M. Huennekens. 1974. Activation of methionine synthetase by a reduced triphosphopyridine nucleotide-dependent flavoprotein system. J. Biol. Chem. 249:6745-53. Goulding, C. W. and R. G. Matthews. 1997. Cobalamin-dependent methionine synthase from Escherichia coli: involvement of zinc in homocysteine activation. Biochemistry 36:15749-57. Goulding, C. W., D. Postigo and R. G. Matthews. 1997. Cobalamin-dependent methionine synthase is a modular protein with distinct regions for binding homocysteine, methyltetrahydrofolate, cobalamin, and adenosylmethionine. Biochemistry 36:8082-91. Gravel, R. A., M. J. Mahoney, F. H. Ruddle and L. E. Rosenberg. 1975. Genetic complementation in heterokaryons of human fibroblasts defective in cobalamin metabolism. Proc. Natl. Acad. Sci. U S A 72:3181-5. Guest, J. R., M. A. Foster and S. Friedman. 1962a. Alternative pathways for methylation of homocysteine by Escherichia coli. Biochem. J. 84:93P-94P.
130 Guest, J. R., E. L. Smith, D. D. Woods and S. Friedman. 1962b. A methyl analogue of cobamide coenzyme in relation to methionine synthesis by bacteria. Nature 195:340-342. Hall, D. A., T. C. Jordan-Starck, R. O. Loo, M. L. Ludwig and R. G. Matthews. 2000. Interaction of flavodoxin with cobalamin-dependent methionine synthase. Biochemistry 39:10711-9. Halpern, J. 1985. Mechanisms of coenzyme B12-dependent rearrangements. Science 227:869-75. Harder, J., R. Eliasson, E. Pontis, M. D. Ballinger and P. Reichard. 1992. Activation of the anaerobic ribonucleotide reductase from Escherichia coli by S-adenosylmethionine. J. Biol. Chem. 267:25548-52. Harlow, E. and D. Lane. 1988. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Havemann, G. D. and T. A. Bobik. 2003. Protein content of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2. J. Bacteriol. 185:5086-95. Havemann, G. D., E. M. Sampson and T. A. Bobik. 2002. PduA is a shell protein of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2. J. Bacteriol. 184:1253-61. Heithoff, D. M., C. P. Conner, U. Hentschel, F. Govantes, P. C. Hanna and M. J. Mahan. 1999. Coordinate intracellular expression of Salmonella genes induced during infection. J. Bacteriol. 181:799-807. Hodgkin, D. C., J. Pickworth, J. H. Robertson, K. N. Trueblood, R. J. Prosen and J. G. White. 1955. Crystal structure of the hexacarboxylic acid derived from B12 and the molecular structure of the vitamin. Nature 176:325-328. Horswill, A. R. and J. C. Escalante-Semerena. 1997. Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8.5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon. J. Bacteriol. 179:928-40. Huennekens, F. M., K. S. Vitols, K. Fujii and D. W. Jacobsen. 1982. Biosynthesis of the cobalamin coenzymes, p. 145-164. In D. Dolphin (ed.), B12, vol. volume 1. John Wiley & Sons, Inc., New York. Jarrett, J. T., D. M. Hoover, M. L. Ludwig and R. G. Matthews. 1998. The mechanism of adenosylmethionine-dependent activation of methionine synthase: a rapid kinetic analysis of intermediates in reductive methylation of cob(II)alamin enzyme. Biochemistry 37:12649-58.
131 Johnson, C. L. V. J., E. Pechonick, S. D. Park, G. D. Havemann, N. A. Leal and T. A. Bobik. 2001. Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene. J. Bacteriol. 183:1577-1584. Jordan, A., E. Pontis, M. Atta, M. Krook, I. Gibert, J. Barbe and P. Reichard. 1994. A second class I ribonucleotide reductase in Enterobacteriaceae: characterization of the Salmonella typhimurium enzyme. Proc. Natl. Acad. Sci. U S A 91:12892-6. Kapadia, C. R. 1995. Vitamin B12 in health and disease: part I--inherited disorders of function, absorption, and transport. Gastroenterologist 3:329-44. Kass, L. 1978. William B. Castle and intrinsic factor. Ann. Intern. Med. 89:983-91. Kessler, D., I. Leibrecht and J. Knappe. 1991. Pyruvate formate-lyase-deactivase and acetyl-CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE. FEBS Lett. 281:59-63. Kofoid, E., C. Rappleye, I. Stojiljkovic and J. R. Roth. 1999. The 17-gene ethanolamine (eut) operon of Salmonella typhimurium encodes five homologues of carboxysome shell proteins. J. Bacteriol. 181:5317-5329. Kolhouse, J. F. and R. H. Allen. 1977. Recognition of two intracellular cobalamin binding proteins and their identification as methylmalonyl-CoA mutase and methionine synthetase. Proc. Natl. Acad. Sci. U S A 74:921-5. Korotkova, N., L. Chistoserdova, V. Kuksa and M. E. Lidstrom. 2002. Glyoxylate regeneration pathway in the methylotroph Methylobacterium extorquens AM1. J. Bacteriol. 184:1750-8. Korotkova, N. and M. E. Lidstrom. 2004. MeaB is a component of the methylmalonyl-CoA mutase complex required for protection of the enzyme from inactivation. J. Biol. Chem. 279:13652-8. Leal, N. A., S. D. Park, P. E. Kima and T. A. Bobik. 2003. Identification of the human and bovine ATP:Cob(I)alamin adenosyltransferase cDNAs based on complementation of a bacterial mutant. J. Biol. Chem. 278:9227-34. Leclerc, D., E. Campeau, P. Goyette, C. E. Adjalla, B. Christensen, M. Ross, P. Eydoux, D. S. Rosenblatt, R. Rozen and R. A. Gravel. 1996. Human methionine synthase: cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders. Hum. Mol. Genet. 5:1867-74. Leclerc, D., M. Odievre, Q. Wu, A. Wilson, J. J. Huizenga, R. Rozen, S. W. Scherer and R. A. Gravel. 1999. Molecular cloning, expression, and physical mapping of the human methionine synthase reductase gene. Gene 240:75-88.
132 Leclerc, D., A. Wilson, R. Dumas, C. Gafuik, D. Song, D. Watkins, H. H. Heng, J. M. Rommens, S. W. Scherer, D. S. Rosenblatt and R. A. Gravel. 1998. Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria. Proc. Natl. Acad. Sci. U S A 95:3059-64. Ledley, F. D., M. R. Lumetta, H. Y. Zoghbi, P. VanTuinen, S. A. Ledbetter and D. H. Ledbetter. 1988. Mapping of human methylmalonyl CoA mutase (MUT) locus on chromosome 6. Am. J. Hum. Genet. 42:839-46. Lenhert, P. G. and D. C. Hodgkin. 1961. Structure of 5,6-dimethylbenzimidazolylcobamide coenzyme. Nature 192:937-938. Ljungdahl, L. and H. Wood. 1982. Acetate biosynthesis. In D. Dolphin (ed.), B12. John Wiley & Sons, New York. Mahoney, M. J., A. C. Hart, V. D. Steen and L. E. Rosenberg. 1975. Methylmalonicacidemia: biochemical heterogeneity in defects of 5'-deoxyadenosylcobalamin synthesis. Proc. Natl. Acad. Sci. USA 72:2799-2803. Mahoney, M. J., L. E. Rosenberg, S. H. Mudd and B. W. Uhlendorf. 1971. Defective metabolism of vitamin B12 in fibroblasts from children with methylmalonic aciduria. Biochem. Biophys. Res. Commun. 44:375-81. Mancia, F., N. H. Keep, A. Nakagawa, P. F. Leadlay, S. McSweeney, B. Rasmussen, P. Bosecke, O. Diat and P. R. Evans. 1996. How coenzyme B12 radicals are generated: the crystal structure of methylmalonyl-coenzyme A mutase at 2 resolution. Structure 4:339-50. Maniatis, T., E. F. Fritsch and J. Sambrook. 1982. Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Marsh, E. N. and C. L. Drennan. 2001. Adenosylcobalamin-dependent isomerases: new insights into structure and mechanism. Curr. Opin. Chem. Biol. 5:499-505. Marsh, E. N., N. McKie, N. K. Davis and P. F. Leadlay. 1989. Cloning and structural characterization of the genes coding for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii. Biochem. J. 260:345-52. Matsui, S. M., M. J. Mahoney and L. E. Rosenberg. 1983. The natural history of the inherited methylmalonic acidemias. N. Engl. J. Med. 293:857-861. Matthews, R. G. 1999. Cobalamin-dependent Methionine Synthase, p. 681-706. In R. Banerjee (ed.), Chemistry and biochemistry of B12. John Wiley & Sons, Inc., New York.
133 Mellman, I., H. F. Willard and L. E. Rosenberg. 1978. Cobalamin binding and cobalamin-dependent enzyme activity in normal and mutant human fibroblasts. J. Clin. Invest. 62:952-60. Mellman, I., H. F. Willard, P. Youngdahl-Turner and L. E. Rosenberg. 1979. Cobalamin coenzyme synthesis in normal and mutant human fibroblasts. Evidence for a processing enzyme activity deficient in cblC cells. J. Biol. Chem. 254:11847-11853. Mellman, I. S., P. Youngdahl-Turner, H. F. Willard and L. E. Rosenberg. 1977. Intracellular binding of radioactive hydroxocobalamin to cobalamin-dependent apoenzymes in rat liver. Proc. Natl. Acad. Sci. U S A 74:916-20. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Miller, V. L. and J. J. Mekalanos. 1988. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170:2575-83. Minot, G. R. and W. P. Murphy. 1926. Treatment of pernicious anemia by a special diet. J. Am. Med. Assoc. 87:470-476. Nordlund, P. and H. Eklund. 1993. Structure and function of the Escherichia coli ribonucleotide reductase protein R2. J. Mol. Biol. 232:123-64. Obradors, N., J. Bada, L. Baldom and J. Aguilar. 1988. Anaerobic metabolism of the L-rhamnose fermentation product 1,2-propanediol in Salmonella typhimurium. J. Bacteriol. 170:2159-2162. Olteanu, H. and R. Banerjee. 2001. Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation. J. Biol. Chem. 276:35558-63. Padmakumar, R. and R. Banerjee. 1995. Evidence from electron paramagnetic resonance spectroscopy of the participation of radical intermediates in the reaction catalyzed by methylmalonyl-coenzyme A mutase. J. Biol. Chem. 270:9295-300. Padmakumar, R. and R. Banerjee. 1997. Evidence that cobalt-carbon bond homolysis is coupled to hydrogen atom abstraction from substrate in methylmalonyl-CoA mutase. Biochemistry 36:3713-8. Palacios, S., V. J. Starai and J. C. Escalante-Semerena. 2003. Propionyl coenzyme A is a common intermediate in the 1,2-propanediol and propionate catabolic pathways needed for expression of the prpBCDE operon during growth of Salmonella enterica on 1,2-propanediol. J. Bacteriol. 185:2802-10.
134 Paschen, S. A. and W. Neupert. 2001. Protein import into mitochondria. IUBMB Life 52:101-12. Pezacka, E. 1993. Identification of and characterization of two enzymes involved in the intracellular metabolism of cobalamin. Cyanocobalamin -ligand transferase and micosomal cob(III)alamin reductase. Biochem. et. Biophys. Acta. 1157:167-177. Pezacka, E., R. Green and D. W. Jacobsen. 1990. Glutathionylcobalamin as an intermediate in the formation of cobalamin coenzymes. Biochem. Biophys. Res. Commun. 169:443-450. Price, G. D., S. Maeda, T. Omata and M. R. Badger. 2002. Modes of active inorganic carbon uptake in the cyanobacterium, Synechococcus sp PCC7942. Functional Plant Biology 29:131-149. Price-Carter, M., J. Tingey, T. A. Bobik and J. R. Roth. 2001. The alternative electron acceptor tetrathionate supports B12-dependent anaerobic growth of Salmonella enterica serovar typhimurium on ethanolamine or 1,2-propanediol. J. Bacteriol. 183:2463-75. Qureshi, A. A., D. S. Rosenblatt and B. A. Cooper. 1994. Inherited disorders of cobalamin metabolism. Crit. Rev. Oncol. Hematol. 17:133-51. Refsum, H., P. M. Ueland, O. Nygard and S. E. Vollset. 1998. Homocysteine and cardiovascular disease. Annu. Rev. Med. 49:31-62. Reichard, P. 1993. The anaerobic ribonucleotide reductase from Escherichia coli. J. Biol. Chem. 268:8383-6. Rickes, E. L., N. G. Brink, F. R. Koniuszy, T. R. Wood and K. Folkers. 1948. Crystalline vitamin B12. Science 107:396-397. Rondon, M. R., R. Kazmierczak and J. C. Escalante-Semerena. 1995. Glutathione is required for maximal transcription of the cobalamin biosynthetic and 1,2-propanediol utilization (cob/pdu) regulon and for the catabolism of ethanolamine, 1,2-propanediol, and propionate in Salmonella typhimurium LT2. J. Bacteriol. 177:5434-9. Rosenblatt, D. S., A. L. Aspler, M. I. Shevell, B. A. Pletcher, W. A. Fenton and M. R. Seashore. 1997. Clinical heterogeneity and prognosis in combined methylmalonic aciduria and homocystinuria (cblC). J. Inherit. Metab. Dis. 20:528-38. Rosenblatt, D. S. and A. B. Cooper. 1990. Inherited disorders of vitamin B12 utilization. Bioessays 12:331-334. Rosenblatt, D. S. and W. A. Fenton. 1999. Inborn errors of cobalamin metabolism, p. 367-384. In R. Banerjee (ed.), Chemistry and Biochemistry of B12. John Wiley & Sons, Inc., New York.
135 Rossi, M., J. P. Glusker, L. Randaccio, M. F. Summers, P. J. Toscano and L. G. Marzilli. 1985. The Structure of a B12 coenzyme: Methylcobalamin Studies by X-ray and NMR Methods. J. Am. Chem. Soc. 107:1729-1738. Roth, J. R., T. A. Bobik and J. G. Lawrence. 1996. Cobalamin (coenzyme B12): synthesis and biological significance. Annual Rev. Microbiol. 50:137-181. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning : a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Saridakis, V., A. Yakunin, X. Xu, P. Anandakumar, M. Pennycooke, J. Gu, F. Cheung, J. M. Lew, R. Sanishvili, A. Joachimiak, C. H. Arrowsmith, D. Christendat and A. M. Edwards. 2004. The structural basis for methylmalonic aciduria. The crystal structure of archaeal ATP:cobalamin adenosyltransferase. J. Biol. Chem. 279:23646-53. Schmieger, H. 1971. A method for detection of phage mutants with altered transducing ability. Mol. Gen. Genet. 110:378-381. Schneider, Z. 1987. The occurence and distribution of corrinoids, p. 157-223. In Z. Schneider and A. Stroinski (ed.), Comprehensive B12. Walter de Gruyter, Berlin. Schneider, Z. 1987. Purification and Estimation of Vitamin B12, p. 111-155. In Z. Schneider and A. Stroinski (ed.), Comprehensive B12. Walter de Gruyter, Berlin. Schneider, Z. and A. Stroinski (eds.). 1987. Comprehensive B12. Walter de Gruyter & Co., Berlin. Schrauzer, G. N. and E. Deutsch. 1969. Reactions of cobalt(I) supernucleophiles. The alkylation of vitamin B12s cobaloximes(I), and related compounds. J. Am. Chem. Soc. 91:3341-50. Schrauzer, G. N., E. Deutsch and R. J. Windgassen. 1968. The nucleophilicity of vitamin B12. J. Am. Chem. Soc. 90:2441-2. Scott, A. I. 2003. Discovering nature's diverse pathways to vitamin B12: a 35-year odyssey. J. Org. Chem. 68:2529-39. Seetharam, B. 1999. Receptor-mediated endocytosis of cobalamin (vitamin B12). Annu. Rev. Nutr. 19:173-95. Segel, I. H. 1976. Biochemical calculations : how to solve mathematical problems in general biochemistry, 2 nd ed. Wiley, New York Shibata, N., J. Masuda, T. Tobimatsu, T. Toraya, K. Suto, Y. Morimoto and N. Yasuoka. 1999. A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase. Structure Fold Des. 7:997-1008.
136 Shively, J. M. and R. S. English. 1991. The carboxysome, a prokaryotic organelle: a mini-review. Can. J. Bot. 69:957-962. Shively, J. M., R. S. English, S. H. Baker and G. C. Cannon. 2001. Carbon cycling: the prokaryotic contribution. Curr. Opin. Microbiol. 4:301-306. Shively, J. M., G. van Keulen and W. G. Meijer. 1998. Something from almost nothing: carbon dioxide fixation in chemoautotrophs. Annu. Rev. Microbiol. 52:191-230. Small, I., N. Peeters, F. Legeai and C. Lurin. 2004. Predotar: A tool for rapidly screening proteomes for N-terminal targeting sequences. Proteomics 4:1581-90. Smith, E. L. and L. F. J. Parker. 1948. Purification of anti-pernicious anaemia factor. Biochem. J. 43:R8-R9. Stabler, S. P. 1999. B12 and Nutrition, p. 343-365. In R. Banerjee (ed.), Chemistry and biochemistry of B12. John Wiley & Sons, Inc., New York. Stojiljkovic, I., A. J. Baumler and F. Heffron. 1995. Ethanolamine Utilization in Salmonella Typhimurium Nucleotide-Sequence, Protein Expression, and Mutational Analysis of the Ccha Cchb Eute Eutj Eutg Euth Gene-Cluster. J. Bacteriol. 177:1357-1366. Suh, S. J. and J. C. Escalante-Semerena. 1993. Cloning, sequencing and overexpression of cobA, which encodes ATP:corrinoid adenosyltransferase in Salmonella typhimurium. Gene 129:93-97. Suh, S.-J. and J. C. Escalante-Semerena. 1995. Purification and initial characterization of the ATP:corrinoid adenosyltransferase encoded by the cobA gene of Salmonella typhimurium. J. Bacteriol. 177:921-925. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin and D. G. Higgins. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24:4876-4882. Toraya, T., S. Honda and S. Fukui. 1979. Fermentation of 1,2-propanediol and 1,2-ethanediol by some genera of Enterobacteriaceae, involving coenzyme B12-dependent diol dehydratase. J. Bacteriol. 139:39-47. Toraya, T. and K. Mori. 1999. A reactivating factor for coenzyme B12-dependent diol dehydratase. J. Biol. Chem. 274:3372-7. Vitols, E., G. A. Walker and R. M. Huennekens. 1966. Enzymatic conversion of vitamin B12s to a cobamide coenzyme, -(5,6-dimethylbenzimidazolyl)deoxyadenosylcobamide (adenosyl-B12). J. Biol. Chem. 241:1455-1461.
137 Vlasie, M., S. Chowdhury and R. Banerjee. 2002. Importance of the histidine ligand to coenzyme B12 in the reaction catalyzed by methylmalonyl-CoA mutase. J. Biol. Chem. 277:18523-7. Vogel, H. J. and D. M. Bonner. 1956. Acetylornithase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218:97-106. Walker, G. A., S. Murphy and F. M. Huennekens. 1969. Enzymatic conversion of vitamin B12a to adenosyl B12: evidence for the existence of two separate reducing systems. Arch. Biochem. Biophys. 134:95-102. Walter, D., M. Ailion and J. Roth. 1997. Genetic characterization of the pdu operon: use of 1,2-propanediol in Salmonella typhimurium. J. Bacteriol. 179:1013-22. Warren, J. W., J. R. Walker, J. R. Roth and E. Altman. 2000. Construction and characterization of a highly regulable expression vector, pLAC11, and its multipurpose derivatives, pLAC22 and pLAC33. Plasmid 44:138-51. Watanabe, F., Y. Nakano, S. Maruno, N. Tachikake, Y. Tamura and S. Kitaoka. 1989. NADHand NADPH-linked aquacobalamin reductases occur in both mitochondrial and microsomal membranes of rat liver. Biochem. Biophys. Res. Commun. 165:675-9. Watanabe, F., Y. Oki, Y. Nakano and S. Kitaoka. 1987. Purification and characterization of aquacobalamin reductase (NADPH) from Euglena gracilis. J. Biol. Chem. 262:11514-8. Watanabe, F., H. Saido, R. Yamaji, K. Miyatake, Y. Isegawa, A. Ito, T. Yubisui, D. S. Rosenblatt and Y. Nakano. 1996. Mitochondrial NADHor NADPH-linked aquacobalamin reductase activity is low in human skin fibroblasts with defects in synthesis of cobalamin coenzymes. J. Nutr. 126:2947-2951. Watkins, D., N. Matiaszuk and D. S. Rosenblatt. 2000. Complementation studies in the cblA class of inborn error of cobalamin metabolism: evidence for interallelic complementation and for a new complementation class (cblH). J. Med. Genet. 37:510-3. Watkins, D. and D. S. Rosenblatt. 1988. Genetic heterogeneity among patients with methylcobalamin deficiency. Definition of two complementation groups, cblE and cblG. J. Clin. Invest. 81:1690-4. Watkins, D. and D. S. Rosenblatt. 1989. Functional methionine synthase deficiency (cblE and cblG): clinical and biochemical heterogeneity. Am. J. Med. Genet. 34:427-34. Watkins, D. and D. S. Rosenblatt. 2001. Cobalamin and inborn errors of cobalamin absorption and metabolism. The Endocrinologist 11:98-104.
138 Watkins, D., M. Ru, H. Y. Hwang, C. D. Kim, A. Murray, N. S. Philip, W. Kim, H. Legakis, T. Wai, J. F. Hilton, B. Ge, C. Dore, A. Hosack, A. Wilson, R. A. Gravel, B. Shane, T. J. Hudson and D. S. Rosenblatt. 2002. Hyperhomocysteinemia due to methionine synthase deficiency, cblG: structure of the MTR gene, genotype diversity, and recognition of a common mutation, P1173L. Am. J. Hum. Genet. 71:143-53. Weissbach, H., A. Peterkofsky, B. G. Redfield and H. Dickerman. 1963. Studies on the Terminal Reaction in the Biosynthesis of Methionine. J. Biol. Chem. 238:3318-24. Weissbach, H., B. Redfield and A. Peterkofsky. 1961. Conversion of vitamin B12 to coenzyme B12 in cell-free extracts of Clostridium tetanomorphum. J. Biol. Chem. 236:PC40-2. Weissbach, H., B. G. Redfield and A. Peterkofsky. 1962. Biosynthesis of the B12 coenzyme: requirements for release of cyanide and spectral changes. J. Biol. Chem. 237:3217-22. Wilson, A., D. Leclerc, D. S. Rosenblatt and R. A. Gravel. 1999. Molecular basis for methionine synthase reductase deficiency in patients belonging to the cblE complementation group of disorders in folate/cobalamin metabolism. Hum. Mol. Genet. 8:2009-16. Zass, R., D. Leupold, M. A. Fernandez and U. Wendel. 1995. Evaluation of prenatal treatment in newborns with cobalamin-responsive methylmalonic acidaemia. J. Inh. Met. Dis. 18:100-101.
BIOGRAPHICAL SKETCH Nicole Aurora Leal was born to Raul and Adrianne Leal, on August 2, 1976, in Santo Domingo, Dominican Republic. Nicole and her family moved to New York City during the summer of 1979; and 2 years later, to South Florida, where she learned English while attending Pinecrest Elementary School. She attended Plantation Key Middle School, and Coral Shores High School, in the Florida Keys. It was during middle school that Nicole first starting taking an active interest in music and science. During her final years of middle school, the tragic loss of her brother Ryan to cancer awakened a desire to study the life sciences. In high school, she continued to pursue both music and science. During this time, Nicole was a member of the honors choir, competed in regional competitions, and was selected to represent her school at choral camp at the University of Miami. She was a key member of the Coral Shores High School Odyssey of the Mind team, where she competed at both the regional and state levels winning the Ranatra Fusca award for creativity in consecutive years. Throughout High School, Nicole volunteered extensively with Youthwish, a nonprofit organization helping children displaced by Hurricane Andrew; and Habitat for Humanity. She earned a degree in microbiology and cell science (with a minor in chemistry) at the University of Florida, in Gainesville, in the Spring of 1999. During this time, she conducted undergraduate research in the laboratory of Dr. Thomas Bobik. In the Summer of 1999, she continued working in Dr. Bobikâ€™s laboratory, studying B12-dependent 1,2-propanediol degradation in Salmonella. She was accepted into Graduate School in the Fall of 1999, and continued 139
140 her work on B12-dependent propanediol degradation, with the successful identification and partial characterization one of the metabolic enzymes involved in this pathway. In the summer of 2001, she switched her focus of study to B12 metabolism in humans, with specific interest in identifying the enzymes involved in AdoCbl formation, which is the basis of this dissertation. She plans to continue her academic study and intellectual development as a postdoctoral fellow.