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Completing the Methionine Salvage Cycle in Bacteria and Plants

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Title:
Completing the Methionine Salvage Cycle in Bacteria and Plants
Creator:
Ellens, Kenneth W
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[Gainesville, Fla.]
Florida
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University of Florida
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1 online resource (103 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Plant Molecular and Cellular Biology
Committee Chair:
HANSON,ANDREW D
Committee Co-Chair:
KLEE,HARRY JOHN
Committee Members:
GREGORY,JESSE F,III
DE CRECY,VALERIE ANNE
PURICH,DANIEL L
Graduation Date:
5/3/2014

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Subjects / Keywords:
Bacteria ( jstor )
Biosynthesis ( jstor )
Comparative genomics ( jstor )
Corn ( jstor )
Enzymes ( jstor )
Genes ( jstor )
Genomes ( jstor )
Genomics ( jstor )
Proteins ( jstor )
Tomatoes ( jstor )
Plant Molecular and Cellular Biology -- Dissertations, Academic -- UF
biochemistry -- metabolism -- omega-amidase -- transaminase
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bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Plant Molecular and Cellular Biology thesis, Ph.D.

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Abstract:
S-Adenosylmethionine is converted enzymatically and nonenzymatically to methylthioadenosine, which is recycled to methionine (Met) via a salvage pathway. In plants and bacteria, enzymes for all steps in this pathway are known except the last: transamination of alpha-ketomethylthiobutyrate to give Met. In mammals, glutamine transaminase K (GTK) and omega-amidase (omega-Am) are thought to act in tandem to execute this step, with GTK forming alpha-ketoglutaramate, which omega-Am hydrolyzes. Comparative genomics indicated that GTK and omega-Am could function likewise in plants and bacteria because genes encoding GTK and omega-Am homologs (i) co-express with the Met salvage gene 5-methylthioribose kinase in Arabidopsis, and (ii) cluster on the chromosome with each other and with Met salvage genes in diverse bacteria. Consistent with this possibility, tomato, maize, and Bacillus subtilis GTK and omega-Am homologs had the predicted activities: GTK was specific for glutamine as amino donor and strongly preferred alpha-ketomethylthiobutyrate as amino acceptor, and omega-Am strongly preferred alpha-ketoglutaramate. Also consistent with this possibility, plant GTK and omega-Am were localized to the cytosol, where the Met salvage pathway resides, as well as to organelles. This multiple targeting was shown to result from use of alternative start codons. In B. subtilis, ablating GTK did not inhibit growth on 5-methylthioribose as sole sulfur source, and ablating omega-Am had only a mild effect. Collectively, these data indicate that while GTK, coupled with omega-Am, is positioned to support significant Met salvage flux in plants and bacteria, it can probably be replaced by other aminotransferases. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: HANSON,ANDREW D.
Local:
Co-adviser: KLEE,HARRY JOHN.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2016-05-31
Statement of Responsibility:
by Kenneth W Ellens.

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Applicable rights reserved.
Embargo Date:
5/31/2016
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907294871 ( OCLC )
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LD1780 2014 ( lcc )

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COMPLETING THE METHIONINE SALVAGE PATHWAY IN BACTERIA AND PLANTS By KENNETH WILLIAM ELLENS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

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2014 Kenneth William Ellens

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Dedicated to my family, Ron, Carol, Ben, Jessica and to my wife Ashley your love, supp ort and sacrifice can be found between the lines of this work.

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4 ACKNOWLEDGMENTS I would like to thank Dr. Robert T. Mullen and Dr. Lynn G.L. Richardson for all mic roscopy experiments and discussions. From the Hanson lab, I thank Michael Ziemak for much technical assistance, Dr. Ocane Frelin for assistance with dual import assays, and other lab member through the years for their friendship and guidance. I thank Dr. A.J.L. Cooper for much biochemical advice and insightful discussion, Dr. Valerie de Crcy Lagard and Dr. W.L. Nicholson for microbiology support, Dr. G. Moorhead for NAGK antibodies, and Dr. K. Cline and his laboratory for advice on organellar import assays. Thanks also to fellow PMCB students Joseph Collins, Cintia Leite Ribeiro, and Yih feng Hsieh for assistance with initial comparative genomic analysis. This project was supported by U.S. National Science Foundation grant number MCB 1153413 ( to A.D.H ). I must also express my gratitude to my committee members for their advice and support. Finally, I especially want to thank my advisor, Andrew Hanson for all the discussions, the insight, and the challenges.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 Centrality of S Adenosylmethionine ................................ ................................ ........ 12 Covalent and Chiral Instability of S Adenosylmethionine ................................ 12 Adomet in Methyl Group Metabolism ................................ ............................... 12 Adomet as A Precursor of Polyamines, Ethylene and Nicotianamine .............. 13 Methionine Salvage ................................ ................................ ................................ 14 Methionine Salvage Discovery in Plants ................................ .......................... 14 Methionine Salvage Cycle in Bacteria ................................ .............................. 15 Methionine Salvage Cycle in Plants ................................ ................................ 16 Completing the Methionine Salvage Cycle Filling the Gap in Knowledge ...... 16 Comparative Genomics ................................ ................................ .......................... 17 Background and Strengths ................................ ................................ ............... 17 The Comparative Genomi cs Approach ................................ ............................ 19 Metabolite Repair ................................ ................................ ................................ .... 21 General Principles ................................ ................................ ............................ 21 Glutamine Transaminase K and Amidase: Metabolite Repair Enzymes ...... 22 2 EVIDENCE THAT GLUTAMINE TRANSAMINASE AND OMEGA AMIDASE POTENTIALLY A CT IN TANDEM TO CLOSE THE METHIONINE SALVAGE CYCLE IN BACTERIA AND PLANTS 1 ................................ ................................ .... 29 Background ................................ ................................ ................................ ............. 29 Results ................................ ................................ ................................ .................... 31 Comparative Genomics ................................ ................................ .................... 31 Am Enzymes ........................... 32 Am Proteins ............................... 34 Growth and Metabolic Phenotypes of B. subtilis Am Disruptants .. 36 Discussion ................................ ................................ ................................ .............. 38 Experimental Procedures ................................ ................................ ........................ 40 Bioinformatics ................................ ................................ ................................ ... 40 Chemicals ................................ ................................ ................................ ......... 41 Am Homologs ................ 41 Enzyme Assays ................................ ................................ ................................ 42

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6 Bacillus subtilis Experiments ................................ ................................ ............ 44 Dual Import Assays ................................ ................................ .......................... 45 Transient Expression and Microscopic Analysis of GFP Fusion Proteins in BY 2 Cells ................................ ................................ ................................ ..... 45 3 GLUTAMINE TRANSAMINA SE K AND OMEGA AMIDASE ANTISENSE LINES IN TOMATO ................................ ................................ ................................ ............ 64 Background ................................ ................................ ................................ ............. 64 The Impact of Met and AdoMet on Growth, Development and Genome Stability ................................ ................................ ................................ .......... 64 Tomato as a Model System ................................ ................................ .............. 65 Design of Antisense Constructs ................................ ................................ ....... 66 Experimental Plan for Transgenic Tomato Lines ................................ .............. 66 Results ................................ ................................ ................................ .................... 67 Expression Analysis of Antisense Tomato Lines ................................ .............. 67 Discussion ................................ ................................ ................................ .............. 68 Experimental Procedures ................................ ................................ ........................ 69 Bioinformatics ................................ ................................ ................................ ... 69 Cloning ................................ ................................ ................................ ............. 70 Transgenic Plants ................................ ................................ ............................. 70 Quantitative PCR ................................ ................................ .............................. 71 4 CONCLUSIONS ................................ ................................ ................................ ..... 77 APPENDIX A ADDITIONAL COMPARATIVE GENOMICS ................................ ........................... 80 B CYCLIZATION OF KETOGLUTARAMATE ................................ ......................... 89 LIST OF REFERENCES ................................ ................................ ............................... 92 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 103

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7 LIST OF TABLES Table page 2 1 K inetic constants of recombinant GTK ketomethylthiobutyrate. ... 47 2 2 Activities of recombinant amidase s agai amide substrates ....... 48 2 3 K inetic constants of recombinant amidase s with ketoglutaramate as substrate ................................ ................................ ................................ ............. 49

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8 LIST OF FIGURES Figure page 1 1 Sites for enzymatic and non enzymatic cleavage of S adenosylmethionine. ..... 24 1 2 Relationship between AdoMet cycle, the Met salvage cy cle and polyamine biosynthesis ................................ ................................ ................................ ........ 25 1 3 The methionine salvage pathway ................................ ................................ ...... 26 1 4 Reaction thought to close the Met salvage cycle in mammals ........................... 27 1 5 Types of associa tions in comparative genomics ................................ ................. 28 2 1 The methionine salvage pathway ................................ ................................ ....... 50 2 2 Comparative genomics of glutamine transam amidase homologs. ................................ ................................ ................................ ........... 51 2 3 Purification of recombinant bacterial and plant Am and GTK proteins. ........... 53 2 4 Analytical size exclusion chromatography of recombinant plant Am and GTK proteins ................................ ................................ ................................ ...... 54 2 5 Substrate preferences of plan t and bacterial GTK proteins. ............................... 55 2 6 Substrate inhibitio n of recombinant GTK enzymes ................................ ............. 56 2 7 N Terminal sequence alignments of plant, human, and bact Am proteins. ................................ ................................ ................................ ....... 57 2 8 In vitro t ranslation and organellar Am and GTK ................... 58 2 9 Representative confocal images of tobacco BY 2 cells transiently expressing Am constructs C terminally fused to GFP ................................ ........... 60 2 10 Representative epifluorescence images of tobacco BY 2 cells transiently expressing tomato GTK constructs with a C ter minal appended Myc tag. ........ 61 2 11 Growth of B. subtilis with 5 methylth ioribose as sole sulfur source. ................... 62 2 12 Synthetic oligonucleotides used in this study. ................................ .................... 63 3 1 Transcript level of G TK in transgenic tomato lines ................................ ............ 72 3 2 Transcript level of A m in transgenic tomato lines. ................................ .......... 74 3 3 Synthetic oligonucleotides used in this study. ................................ .................... 76

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9 A 1 Arabidopsis genes c Am. ................................ ....... 85 A 2 Connection of arginine (Arg) biosynthesis to GTK and A m pathway. ............. 86 A 3 DA P lysine biosynthesis pathway ................................ ................................ ...... 87 A 4 Phylogenetic analysis of GTK and variants of diaminopi melate aminotransferases. ................................ ................................ ............................. 88 B 1 The fa ketoglutaramate (KGM). ................................ ................................ 90 B 2 Amidase ( Am) enzy me quantity validation assays. ................................ ..... 91

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10 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 COMPLETING THE METHIONINE SALVAGE PATHWAY IN BACTERIA AND PLANTS By Kenneth William Ellens M ay 2014 Chair: Andrew D. Hanson Major: Plant Molecular and Cellular Biology (PMCB) S Adenosylmethionine is converted enzymatically and nonenzymatically to methy lthioadenosine, which is recycled to methionine (Met) via a salvage pathway. In plants and bacteria, enzymes for all steps in this pathway are known except the last: ketomethylthiobutyrate to give Met. In mammals, glutamine transaminase Am) are thought to act in tandem to execute Am hydrolyzes. Comparative Am could function likewise in plants and bacteria because genes encodi Am homologs (i) co express with the Met salvage gene 5 methylthioribose kinase in Arabidopsis and (ii) cluster on the chromosome with each other and with Met salvage genes in diverse bacteria. Consistent with this possibility, tomato, maize, and Bacillus subtilis Am homologs had the predicted activities: GTK was specific for glutamine as amino donor and strongly ketoglutaramate. Also consistent with t Am were localized to the cytosol, where the Met salvage pathway resides, as well as to organelles. This multiple targeting was shown to result from use of alternative start codons. In B. subtilis ablating GTK did not inhib it growth on 5 methylthioribose as sole

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11 Am had only a mild effect. Collectively, these data indicate Am, is positioned to support significant Met salvage flux in plants and bacteria, it can proba bly be replaced by other aminotransferases.

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12 CHAPTER 1 INTRODUCTION Centrality of S A d en o sylm et hionine Covalent and Chiral I nstability of S Adenosylmethionine S A denosylmethionine (AdoMet) is an integral metabolite in the biological infrastructure in eukaryotic and prokaryotic organisms. AdoMet is the major substrate for methyltransferase reactions which methylate primary and secondary metabolites, lipids, cell wall polymers as well as nucl eic acids and histones (Roj e, 2006; Sauter et al., 2013). The chemical properties of AdoMet, particularly the fact that this compound is highly labile have been well documented (Eloranta and K ajander 1984; Hoffman, 1986). Enzymatic as well as pH depend ent chemical cleavage points are identified in Figure 1 1. Some of the spontaneous cleavage products have been found to inhibit other enzymatic reactions (Eloranta and K ajander 1984; Hoffman, 1986). On e particular example can be found in the case of spo ntaneous cleavage point III (Figure 1 1) where the products are homoserine lactone and methylthioadenosine (MTA) ; the latter compound acts as an inhibitor of polyamine synthase reactions (Parveen and Cornell, 2011 ). In addition to the litany of covalent cleavage points present on AdoMet spontaneous racemization from the biologically active ( S,S ) AdoMet to the inactive ( R,S ) form has also been reported (Hoffman 1986). The ( R,S ) form of AdoMet is known to strongly inhibit the ethylene synthesis enzyme ACC synthase (Satoh and Yang 1989) as well as a n array of methyltransferases ( Borchardt and Wu 1976 ). Adomet in Methyl Group Metabolism Methyl transfer reactions are central to cellular biochemistry and AdoMet is undoubtedly the most commonly used methyl do nor. The methyl group that is bound to

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13 the charged sulfur atom of AdoMet (Figure 1 1) is very reactive toward polarizabl e nucleophiles (N, O and S) (Cheng and Roberts, 2001). Methyltransferases that rely upon AdoMet as methyl donor have a vast array of t argets such as DNA, RNA, proteins, lipids, polysaccharides, and many small molecules. Some methyltransferases have been found to demonstrate strict specificity for a single substrate while many others accept a broad range of substrates. In plants a subs tantial portion of the carbon flux through AdoMet as a methyl donor ultimately is incorporated into lignin ( Boerjan, 2003 ; Whetten and Sederoff 1995). Aside from the regulatory roles served by protein and DNA methyltransferases, these enzymes are also re sponsible for the biosynthesis of various plant metabolites. An example which ties into lipid metabolism is the conversion of phosphoethanolamine to phosphocholine performed by phosphoethanolamine N methyltransferase. Phosphocholine is the precursor to c holine (Nuccio et al., 2000) which is used to synthesize the phospholipid phosphatidylcholine ; this compound comp ri ses about 40 60% of lipids in non plastid membranes ( Bolognese and McGraw 2000 ). In plants and animals, the organization of chromatin structure is great ly affect ed by the methylation patterns of histone proteins and DNA (Roje, 2006). Adomet as A Precursor of Polyamines, Ethylene and Nicotianamine The biosynthesis of ethylene, polyami nes (PA), and nicotianamine (NA) all rely upon AdoMet as a precursor (Figure 1 2 ). Ethylene is involved in regulation of growth, development, and responses to stress and pathogen attack in plants (Bleecker and Kende, 2000). ACC synthase produces 1 aminoc yclopropane 1 carboxylic acid (ACC) and the by product MTA. This reaction is also the rate limiting step of ethylene biosynthesis, and is followed by the conversion of ACC to ethylene by ACC oxidase (Bleecker and Kende, 2000). The polyamine pathway begins with the decarboxylation of

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14 AdoMet by AdoMet decarboxylase which supplies the amin opropyl moiety for PA synthesis and is the rate limiting step of this biosynthetic pathway (Mattoo et al. 2010). With the simple diamine putrescine as the starting point, spermidine synthase adds an aminopropyl moiety to generate the triamine spermidine and the tetraamine spermine is formed after a successive reaction, this time by spermine synthase (Figure 1 2) After each reaction the by product MTA is formed PAs are found in all living organisms and play roles in development and stress responses. PAs are positively charged at physiological pH, and are known to chemically interact with DNA, RNA, phospholipids, and some proteins (Roje, 2006; Sauter et al., 2013 ). NA is an essential chelator for the intracellular transport and delivery of iron and other metals used by metal binding proteins. Three AdoMet units are used by NA synthase which is responsible for the biosynthesis of NA once again MTA is the common b y product of this reaction (Roje, 2006 ; Van de Poel et al., 2013 ) Methionine Salvage Methionine Salvage Discovery in Plants The study of ethylene synthesis was of great importance to the discovery of a recycling pathway of Met. The connection between Met and ethylene was first strongly established by Lieberman et al. (Lieberman et al., 1966) who demonstrated a significant increase in ethylene production after apple slices were incubated with Met. This work implicated Met as the precursor to ethylene in a pple tissue. In 1972, Baur and Yang were also studying ethylene production in apple fruit and observed that there was only enough Met for a few hours of ethylene production. However, it was known that stored apples produced ethylene for months, and durin g this time no volatile sulfur compounds

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15 were released by the fruit. This led to the postulation that the sulfur was being recycled to replenish the Met pool (Baur and Yang, 1972). Methionine Salvage Cycle in Bacteria In bacteria MTA is converted in 8 st eps (Figure 1 3) although some variations are present where two steps may be accomplished with one enzyme. The salvage pathway begins with mtnN an MTA nucleosidase that hydrolyzes MTA to methylthioribose (MTR) and adenine. MTR is then phosphorylated b y MTR kinase ( mtnK) to form 5 methylthioribose 1 phosphate (MTR 1 P). In some organisms, commonly in facultative anaerobes living on rich media, MTR can be excreted from the cell (Sekowska et al., 2004). An alternative step has been identified where MTR 1 P is produced directly from MTA by MTA phosphorylase ( mtnP ). The ribose ring is then opened by methylthioribose 1 phosphate isomerase ( mtnA ) to produce methylthioribulose 1 phosphate (MTRu 1 P). A dehydration reaction performed by MTRu 1 P dehydratase ( mtnB ) yields 2,3 diketo 5 methylthiopentyl 1 phosphate (DKP 1 P). The diketone is further metabolized to 2 hydroxy 3 keto 5 methylthiopentenyl 1 phosphate (HKMP 1 P) by 2,3 diketo 5 methylthiopentyl 1 phosphate enolase ( mtnW ). This step is followed by 2 hydroxy 3 keto 5 methylthiopentenyl 1 phosphate phosphatase ( mtnX ) which produces 1,2 dihydroxy 3 keto 5 methylthiopentene (DHKMP). In some bacteria, Klebsiella pneumoniae for example, there is a single enolase phosphatase ( mtnC ) which is responsible for the production of DHKMP. An acireductone dioxygenase ( mtnD ) then produces 2 keto 4 methylthiobutryate (KMTB) keto acid of Met. The final step in the pathway is a transamination ; it has generally been thought that several aminotransferase s can mediate this step and that they can all more or less replace one another (Sekowska et al., 2004).

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16 Methionine Salvage Cycle in Plants In plants, the pathway begins the same as the first variant in bacteria with an MTA nucleosidase (MTN) of which there are two homologs in Arabidopsis (MTN1 [At4g38800] and MTN2 [At4g34840]) (Rzewuski et al., 2007). One MTR kinase has been found in Arabidopsis to complete the second reaction (MTK1, At1g49820) that produces MTR 1 P (Sauter et al., 2004). There has also been o nly one isomerase identified to produce MTRu 1 P (MTI, At2g05830 (Pommerrenig et al., 2011). Interestingly, in Arabidopsis there is a single (multi domain) protein that performs the dehydratase, enolase and phosphatase reactions called DEP1 (At5g53850) (P ommerrenig et al., 2011). Four homologous sequences have been identified in Arabidopsis where an acireductone oxygenase step produces KMTB (ARD1 [At4g14716], ARD2 [At4g14710], ARD3 [At2g26400], and ARD4 [At5g43850]) (Pommerrenig et al., 2011, Sauter et al ., 2005). The final step of the pathway is also ambiguous in plants, with a range of aminotransferases being cited as potential candidates. A recent report used BLAST searches with bacterial sequences as the query protein to return results which ranged f rom aspartate aminotransferases to branched chain aminotransferases (Pommerrenig et al., 2011 ; Sauter et al., 2013 ). C ompleting the Methionine Salvage Cycle Filling the Gap in Knowledge As noted above, a specific enzyme has not been definitively assigned to complete t he fina l step of the Met salvage cycle. Evidence from assays of cell extracts and of recombinant aminotransferases suggest that perhaps even several general transaminases could be responsible (Albers, 2009; Berger et al., 2003; Pirkov et al. 2008; Pommerrenig et al., 2011; Sekowska et al., 2004). The intrinsic problem with using a general transaminase is that the transamination reaction is readily reversible

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17 ( Taylor et al., 1998) and KMTB is known to be chemically unstable (Gao et al., 1998) Some evidence in mammals has been found for a specific type of transaminase. In one report, the researchers were selecting transaminases that transfer the amino group from position on account of the stereochemistry at the 2 keto position of KMTB ( Pirkov et al., 2008) It was also known from experiments in mammals that a major source of amine nitrogen of Met is glutamine (Backlund et al., 1982) Specifically, a glutamine dependent transaminase, glutamine transaminase K (GTK ) [E.C. 2.6.1.64] has been proposed to be involved in the transamination of KMTB to Met (Cooper, 2004) Such ketoglutara mate ( KGM), which is amidase Am) ketoglutarate and ammonia. Furt her biochemical evidence implicates the nitrilase like protein Nit2 as an Am [E.C. 3.5.1.3] that hydrolyzes KGM and so limits its accumulation (Krasnikov et al., 2009) In keto acid of Met, is the preferred substrate of GTK (Cooper, 2004) One possible rationale for using a glutamine dependent transaminase for this reaction is keto acid produced, KGM, cyclizes spontaneously and rapidly (Hersh, 1971) As with hydrolysis, cyclization remove s KGM from the system, thereby pul ling the transamination in the direction of Met formation (Figure 1 4 ). There are thus Am could potentially mediate KMTB transamination in bacteria a nd plants as well as mammals closing the Met salvage cycle. Comparative Genomic s Background and Strengths Traditional methods for identification of novel enzymes and pathways involved the isolation of mutants or protein purification from large quantities of the organism of interest (Bensen et al., 1995 ; Chapple et al., 1990). Today, genomic resources allow for

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18 in silico evidence to be accumulated before any bench work beg i n s This process of making and validating functional predictions is now one of the additional tools at the disposal of the experimen tal scientist (Naponelli et al., 2008 ; Eudes et al., 2008 ; Haas and de Crcy Lagard, 2009). The formerly stringent definition of a gene function include s an experimentally defined role supported by a molecular and a biological dimension. The molecular d imension for a given enzyme is established when its catalytic reaction is elucidated whereas the discovery of the pathway in which the enzyme participates fulfils the biological dimension (Blaby Haas and de Crcy Lagard, 2011). Therefore, until both dimen Sequence similarity is often used as a benchmark to project experimentally established functions of proteins from one species to another (Osterman and Overbeek, 2003).These homology based methods h ave been used effectively in the past (Emanuelli et al., 2003 ; Harper and Bar Peled, 2002), however they can also result in imprecise or even incorrect annotations, such as placing an unknown protein in a general cla ss (Hanson et al., 2009). Determining a precise function requires approaches that extend beyond homology. This is where comparative genomics, as a tool, can help establish the link between gene and function. Comparative genomics is the integration of variou s types of genomic and post genomic evidence (Hanson et al., 2009), as summarized in Fig 1 5 The guilt by association principle is readily applied in comparative genomics, where an unknown gene is found associated with (by genomic and/or post genomic ev idence ) known genes allowing for the function of the unknown gene to be inferred from these associations (Aravind, 2000) Gene clustering is found

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19 commonly in prokaryotes, where functionally related genes often occur in operons. There are also cases wher e genes are divergently transcribed or genes will be found consistently in close proximity to one another (Overbeek et al., 1999). Gene fusions can imply functional relatedness as two individual genes with their own respective activities are found joined together (Enright and Ouzounis, 2001) Genes that are involved in a common pathway or process are often controlled by the same regulatory site which may be a DNA motif or riboswitch ( Gelfand et al., 2000) Phlylogenetic occurrence is based on the assumptio n that proteins involved in the same pathway will tend to either be preserved or eliminated in a new species during evolution (Pellegrini et al., 1999). Functional predictions can also be made from post genomic evidence w ith the use of co expression data f rom microarrays (Obayashi et al., 2011), and more recently RNA seq (Wang et al., 2009) The emerging protein protein interaction databases (Salwinski et al. 2004, Bickerton et al., 2011) can assist with revealing associations along with organelle proteome s and subcellular location databases (Heazlewood et al., 2007). Gene essentiality data from knockout collections allows for a phenotype to be connected to a particular gene or even determine if the gene is vital to the organism (Gerdes et al., 2006). Str ucture databases can assign a protein of unknown function to a general protein class when homology based analysis may not have been useful (Lee et al., 2011). The Comparative Genomics Approach The principles stated above are used during the comparative g enomics approach that was outlined by Osterman and Overbeek (2003). There are three major steps that are implemented i n a missing gene study, (i) building a case, (ii) evidence accumulation

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20 and analysis and (iii) experimental verification. Establishing a functional context is the This is done by gaining perspective on the known genes and the metabolites involved in the pathway of interest. The public web resources and databases, (i.e. KEGG [ http://www.genome.jp/kegg/ ], BRENDA [ http://www.brenda enzymes.org/ ] Expasy [ http://ca.expasy.org/ ] ) in addition to literature and other published biochemical information, are the main resources available to assist in this first step. The inventory of the functional roles that were identified is then added to the rows of a table whereas the columns contain a list of genomes. It is important to note that t he presence of a functional role for a specific o rganism is inferred by homology and/or the propagation of an annotation. There is signment can be vague or wrong, w hich is why multiple forms of genomic and post genomic evidence are used in the evidence accumulation stage. From the table, specific variants of the pathway can be observed in different organisms and can allow for identification of currently miss ing genes. There are limitations to this method however, such as enzymes with broad specificities or non committed enzymes that may take the place of a missing gene (Osterman and Overbeek, 2003) It is during the evidence accumulation step that some filt ers can be applied to discard a candidate, if the evidence is lacking. The genomic and post genomic evidence presented in Fig ure 1 5 is now used to infer functional coupling to create a short list of candidate genes. In order for a particular candidate g ene to be fully characterized both its molecular and biological dimensions must have experimental evidence. Briefly, the molecular dimension can be supported by enzymatic assays using recombinant protein of the gene of interest

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21 performing the predicted r eaction. The biological dimension is supported when a phenotype is returned to wild type by the complementation of a mutant with the ablated gene itself and /or homologs from different organisms. Often the list of candidate genes is quite short and the va lidation process can be efficiently implemented to attain results to characterize the missing gene (Osterman and Overbeek, 2003). Metabolite Repair General Principles made thousands of complete genomes available as a resource to the scientific community. The issues discu ssed above relating to homology based annotation methods are confounded because of the f act that even well studied genomes have a large proportion of their genes without an associated function. This is particularly true for eukaryotic genomes, in which often more than 50% of the genes are not assigned function s (Gerdes et al., 2011) Of these genes of unknown function, many are predicted to encode protein s ar e widely conserved across kingdoms (Galperin and Koonin, 2004; Galperin and Koonin, 2010). chemical biology increasingly recognizes that enzymes can form useless or toxic side p roducts ( Linster et al., 2013; Casadess, 1998). Evidence also exists that metabolites are damaged purely by chemical reactions (Golubev, 1996) It seems that enzyme mistakes and chemical damage are anci ent metabolic problems and the evolutionary driving force for metabolite repair systems (Galperin et al., 2006; Linster et al., 2013) These metabolite repair systems involve numerous underrecognized enzymes acting on enzymatic

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22 mistakes and spontaneous ch emical breakdown products. Repair enzymes are widely conserved because the enzymatic errors and chemistry of the damage are the same across all organisms (Vinci and Clarke, 2010). One of the possible reasons that many of the repair enzymes have largely g one undetected is that they affect fitness not survival. Only in times of stress w ould an organism require these repair reactions to ensure the integrity of metabolism. In fact, metabolite damage appears to be so ubiquitous and pervasive that it has been postulated to be associated with aging (Gladyshev, 2014). Glutamine Transaminase K and Amidase: Metabolite Repair Enzymes The first compound in the Met salvage pathway is MTA, which is the by product of the biosynthesis of ethylene, polyamines, and nicot ianamine (Figure 1 2) as well as chemical breakdown of AdoMet (Hoffman, 1986; Roje, 2006). Already in a minor capacity, by recycling MTA to Met (Figure 1 3), this salvage pathway can be categorized as a repair system What strengthens the case of the Me t salvage cycle as a repair system is the fact that KMTB which is transaminated to produce Met is chemically unstable and thus its accumulation must minimized (Gao et al., 1998). Corroborating this view are reports in mammals of a specific glutamine depe ndent transaminase, GTK, which has a high affinity for KMTB (Cooper, 2004). What makes keto acid, KGM, which is produced during the formation of Met spontaneously cyclizes and is the substrate for an Am (Figure 1 4). T ogether these two phenomena cause this reaction to be essentially irreversible, thus mi nimizing KMTB build up. In k eeping with this theme, KGM is also known to become toxic at elevated concentrations ( Vergara Am as a repair enzyme as well. Taken together, biochemical logic therefore points to the

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23 GT K A m duo as the KMTB transamination syst em that detoxifies problematic metabolites in parallel with regenerating Met from MTA.

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24 Figure 1 1. Sites for enzym at ic and non enzym at ic cleavage of S adenosylmethionine Sites I, II, III and I V are subject to pH dependent non enzym at ic cleavage. S ite IV is also cleaved by a variety of methyltransferases and Site V is attacked by S adenosylmethionine decarboxylase (Eloranta et al., 1984; Hoffman 1986)

PAGE 25

25 Figure 1 2 Relationship between AdoMet cycle, the Met salvage cycle and polyamine biosynthesis. dcAdoMet is generated from AdoMet by the action of AdoMetDC. AdoMet is also required for ethylene and NA biosynthesis (black arrows). Met is salvaged from MTA produced from PA, ethylene and NA bios ynthesis (orange line). In the A do M et cycle (bl ue arrows), AdoMet i s synthesized from Met by AdoMetS. S Adenosylhomocysteine (SAH) is generated from AdoMet through donation of the methyl group catalyzed by one of hundreds of methyltransferases. SAH is hydroly z ed to homocysteine, which is converted into Met by MS. Enzymes are indicated in light orange boxes. Figure adapted from Sauter et al., 2013.

PAGE 26

26 Figure 1 3. The methionine salvage pathway. ( A ) Steps in the Met salvage pathways in bacteria (gene names in italics) and plants (gene names in green ). Alternative bacterial steps are shown as gray arrows. Abbreviations: MTA, methylthioadenosine; MTR, methylthioribose; MTR 1 P, 5 methylthioribose 1 phosphate; MTRu 1 P, methylthioribulose 1 phosphate; DKP 1 P, 2,3 diketo 5 methylthiopentyl 1 phosphate; HKMP 1 P, 2 hydroxy 3 ke to 5 methylthiopentenyl 1 phosphate; DHKMP, 1,2 dihydroxy 3 keto 5 ketomethylthiobutryate. The final step to close the salvage cycle is performed by an unknown transaminase.

PAGE 27

27 Figure 1 4 Reaction thought to close the Met salvage cycle in mammals. Glutamine dependent transamination by glutamine transaminase K (GTK) in concert with ketoglutaramate spontaneously cyclizes to 5 hydroxy 2 oxoproline and that a t neutral pH the equilibrium favors the ring form (99.7%) over the open chain form (0.3%) (Cooper, 2004).

PAGE 28

28 Figure 1 5 Types of associations in comparative genomics. Multiple types of evidence gathered from genomic and post genomic resources are integrated in order to make predictions on gene function. Figure retrieved from Hanson et al., 2009

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29 CHAPTER 2 1 EVIDENCE THAT GLUTAMINE TRANSAMINASE AND OMEGA AMIDASE POTENTIALLY ACT IN TANDEM TO CLOSE THE METHIONINE SALVAGE C YCLE IN BACTERIA AND PLANTS 1 Background The methionine (Met) salvage pathway also called the Yang cycle occurs in bacteria, animals, and plants (Albers, 2009; Miyazaki and Yang, 1987; Sekowska et al., 2004). This pathway recycles Met from 5' methylthi oadenosine (MTA), which is formed from S adenosylmethionine (AdoMet) as a by product of the synthesis of polyamines, ethylene, and other compounds (Albers, 2009), and also comes from spontaneous AdoMet breakdown (Hoffman, 1986). In essence, the pathway, wh ich has several variants, rebuilds Met from the methylthio and ribose moieties of MTA plus a transamination derived amino group (Fig. 2 1A). For bacteria and plants, enzymes and genes are well defined for all the steps in ketomethylthiobutyrate (KMTB) to Met (Pommerrenig et al., 2011; Sekowska et al., 2004). Assays of cell extr acts and of recombinant aminotransferases suggest that several enzymes could be responsible (Albers, 2009; Berger et al., 2003; Pirkov et al., 2008; Pommerrenig et al., 2011; Sekowska et al., 2004). However, some of these studies did not test all potential amino donors (most notably omitting glutamine and asparagine) and none of them attempted to show which if any of the measured activities carry the bulk of the salvage flux in vivo 1 Original publication: Ellens, K.W., Richardson, L.G.L., Frelin, O., Collins, J., Ribeiro, C.L., Hsieh, Y. f., Mullen, R.T., Hanson, A.D., 2014. Evidence that glutamine transaminase and omega amidase can act in tandem to close the methionine salvage cycle i n bacteria and plants. Phytochemistry. Accepted. 2014 by Elsevier

PAGE 30

30 The KMTB transamination step is somew hat better characterized in mam malia n tissues, where enzymological (Backlund et al., 1982; Cooper, 2004) and in vivo 15 N tracer (Hoskin et al., 2001) data implicate a distinctive glutamine dependent aminotransferase, glutamine transaminase K (GTK) [E.C. 2.6.1.64]. The glutamine transaminatio ketoglutaramate (KGM), is potentially toxic but is removed by ketoglutarate and ammonia (Cooper, 2004; Jaisson et al., 2009; Krasnikov et al., 2009) (Fig. 2 1B). In this the direction of Met formation and so lowers KMTB levels (Cooper, 2004). As KMTB is unstable and yields toxic products (Cooper, 2004; Gao et al., 1998), keeping its level low may be benefic ial. Am that respectively Am activity (Berger et al., 2003; Cobzaru et Am homologs (Gerdes et al., 2011; Hudson et al., 2006). Moreover, the activities of both enzymes have been detected in plants (Huang and Ireland, 1991; Lloyd and Joy, 1978; Streeter, ketosuccinamate (the transamination product o f asparagine) rather than KGM (Lloyd and Joy, 1978; Streeter, 1977). There Am could potentially mediate KMTB transamination in bacteria and plants as well as in mammals. The importance of Met salvage in bacteria and plan ts (Miyazaki and Yang, 1987; Sekowska et al., 2004) led us to revisit the enigmatic KMTB transamination step, beginning with comparative genomics and transcriptomics analyses to identify

PAGE 31

31 candidate aminotransferase genes. Such analyses have a strong track r ecord in identifying genes encoding metabolic functions in prokaryotes and plants (Hanson et al., 2009). At the outset, we predicted that plant KMTB aminotransferase genes would encode cytosolic proteins because other plant Met salvage enzymes lack obvious targeting signals and so are most probably cytosolic (Sauter et al., 2004; Sauter et al., 2005). Having obtained comparative genomic evidence associating bacterial and plant Am genes with Met salvage, we characterized representative plant and ba Am proteins localize both to the cytosol and to organelles. We also reinvestigated the effects of inactivating the B. subtilis Am genes, earlier work on this (Sekowska and Danchin, 2002) being inconclusive. Re sults Comparative Genomics Genes encoding GTK and Am homologs (henceforth GTK, Am ) are adjacent in the Bacillus subtilis genome and flanked by Met salvage genes (Sekowska et al., 2004). These arrangements point to possible functional relationships between GTK and Am and between these enzymes and Met salvage. To assess the prevalence of such arrangements, we used the SEED database and its tools (Overbeek et al., 2005) to compare the distribution and chromosomal clustering of GTK, Am, and Met salvage genes in 588 representative bacterial genomes. Of these genomes, 426 (72%) encode Am and 204 (35%) encode GTK. Of the 204 with GTK, 202 have Am and only two do not. Thus, GTK almost never occurs without Am whereas Am often occu rs without GTK (Figs. 2 2A and 2 2B). Moreover, when GTK and Am genes are present, they are often neighbors (Figs. 2 2B and 2 2C). These distribution and

PAGE 32

32 clustering data suggest that GTK action requires Am but that Am can act independently of GTK. T he occurrence of a Met salvage pathway was assessed by the presence of genes encoding m ethylthioribose 1 phosphate isomerase ( mtnA ) and acireductone dioxygenase ( mtnD ) the two enzymes common to all variants of the pathway (Fig. 2 1A). Of 82 genomes having mtnA and mtnD 64 also have GTK and Am and in 28 of these cases GTK and Am cluster with the Met salvage genes (Figs. 2 2A, 2 2B, and 2 2C). However, of the 82 gen omes with mtnA and mtnD 16 lack GTK (Fig. 2 2B). These patter ns indicate (i) that GTK and Am could potentially close the Met salvage cycle in a majority (78%) of the bacteria surveyed; (ii) that in other bacteria, a different enzyme(s) must close the cycle, and (iii) that because 202 genomes have GTK and Am and only 64 also have Met salvage, GTK and Am must have other, widely occurring roles besides Met salvage. Possible associations between GTK, Am and Met salvage in plants were probed using the co ex pression tools for Arabidopsis genes in the ATTED II da tabase (Obayashi et al., 2011) Consistent with such an association, the GTK and Am genes occurred together in a network with the Met salvage gene 5 methylthioribose kinase, as well as genes related to glutamate or aspartate metabolism (Fig. 2 2D). Char acterization o f Recombinant GTK a nd Am Enzymes The tomato and maize GTKs ( Solyc11g013170 and GRMZM2G067265 ) were expressed as His tagged N terminal Nus fusions, and further purified by Ni 2+ affinity chromato graphy, followed by a size exclusion step to remove degr adation products ( Fig. 2 3 A). T Am protein of tomato ( Solyc08g062190 ), the two Am proteins of

PAGE 33

33 maize ( GRMZM2G169365 and GRMZM2G156486 ), B. subtilis Am (MtnU), and B. subtilis GTK (MtnV) were expressed in E. coli as C terminally His tagged proteins and purified by Ni 2+ affinity chromato graphy (Fig. 2 3 B ). B. subtilis Am lost activity during this procedure, and was consequently studied in a d esalted total protein extract ( Fig. 2 3 B). Analytical size exclusion chromatography was used to de ter mine the oligomeric state of the affinity purified proteins. The plant GTK Nus fusions B. subtilis GTK, and the active form of each Am b ehaved as dimers ( Fig. 2 4 ), as do mammalian GTK and Am ( Cooper and Meister, 1974; Krasnikov et al., 2009). With KMTB as amino acceptor, the plant and bacterial GTKs were almost completely specific for glutamine as amino donor, other amino acids giving no more than 1% of the activity measured with glutamine (except for tomato GTK with histidine, where activity w as 5% of that with glutamine) (Fig 2 5 A). This finding for the B. subtilis enzyme agrees with a previous report (Berger et al., 2003) With glutamine as amino ketoisocaproate, and keto methylvalerate ( Fig. 2 5 B ). Upon investigation of the kinetic properties, substrate inhibition by KMTB was observed for all three GTKs; there appear to be no previous reports of this phenomenon for any GTK. Kinetic parameters were estimated by non linear regression curve fitting (Copeland, 2000; Winge et al., 2008) (Fig. 2 6 ). The K m values for all three enzymes for KMTB were within two orders of magnitude of each other (Table 2 1) and of that of mammalian GTK (0.92 mM) (Cooper and Meister, 1974). The K i values were also quite similar to one another. The k cat value for B. subtilis GTK

PAGE 34

34 was quite similar to mammalian GTK (3.2 s 1 ) (Cooper and Meister, 1974) but those for the plant GTKs we re an order of magnitude lower. These low values may reflect the influence of the Nus tag, which was found to be essential for solubility The three plant Am proteins showed far high er activity against KGM than against ketosuccinamate (the transamination product of asparagine) and somewhat higher activity than against succinamate (an artificial substrate) (Table 2 2). All three proteins showed little or no activity against glutamine, asparagine, or the aliphatic amide n butyramide (Tab le 2 2). The order of magnitude difference between the activities with KGM and ketosuccinamate parallels the differences reported for mammalian Am s (Jaisson et al., 2009; Krasnikov et al., 2009). The K m and k cat values for the plant enzymes were very s imilar to each other and to those of mammalian enzyme s (Table 2 3) (Jaisson et al., 2009; Krasnikov et al., 2009) The B. subtilis Am could not be characterized in detail because it could not be purified in active form. Subcellular Localization of Plant Am P roteins Relativ e to their bacterial and mammalian homologs, plant GTK Am proteins have N termi nal extensions; these extensions show little sequence conservation and are generally predicted to be plastid and/or mitochondri al targeting pe ptides Alig ning the N terminal regions of both proteins from angiosperms, gymnosperms, and mosses showed that a Met residue was always present in a six Am) or 26 residue (GTK) window just upstream of where the mature protein probably begins (Fig. 2 7 ) This ob servation shows that each protein has a potential alternative start site that, if used, would eliminate the targeting peptide. To study the possible second start sites Am and GTK constructs were used

PAGE 35

35 as templates for in vitro transcript ion/trans lation reactions: (i) native full length cDNAs, (ii) full length cDNAs with the putative al ternative start Met changed to l eu cine (M2L), and (iii) truncated cDNAs (T) beginning at the putative alternate start (Fig. 2 8 A). I n the Am, the native full length template produced two proteins in similar amounts. The full length M2L template yielded essentially only the larger protein, whereas the truncated T template gave only the smaller one. Results for GTK were similar : the full length native template again gave two proteins, the M2L template increased albeit slightly, the proportion of the larger protein, and the truncated T template gave only the smaller one. These findings indicate that both proteins indeed have a s econd start site, at least in vitro That chang ing the second site in GTK to leucine (TTG) did not more ful ly suppress initiation at this site indicate s th at TTG, at least at this position (i.e. context) in the GTK open reading frame, can serve as a start cod on. The use of TTG as a start codon is not unprecedented in plants (Gordon et al., 1992) Am and GTK was invest igated by dual import assays (Rudhe et al., 2002) in which the radiolabeled proteins and positive controls for mitochondrial and chloroplast import, were incubated with mixtures of purified pea chloroplasts and mito chondria, and thereafter, organelles were incubated (or not) with thermolysin to remove any non imported proteins and repurified In assays Am and GTK ( whose sec ond start Mets were changed to leucine) chloroplasts contained a labeled product that was smaller than the full length precursor and thermolysin resistant, as expected for a translocated protein (Fig. 2 8 B Am, the mitochondria contained very little thermolysin resistant product. For GTK, however, faint but clear mitochondr ial bands corresponding to the original translation products survived

PAGE 36

36 thermolysin treatment, suggesting possible protein import w ithout subsequent processing, or with slow processing (Fig. 2 8 B ). Because in vitro import data ca nnot demonstrate cytosolic localization, the possibility of cytosolic as well as organellar localization was explored using tobacco BY 2 cells transiently ex Am and GTK constructs fused at their C termini to a monomeric version of the green fluorescent protein (GFP) or to the M yc epitope. Am protein without its N terminal targeting sequence localized to the cytosol (Fi g. 2 9 ). The native full Am construct however, appeared in both the cytosol and plastids, as did a construct consisting of GFP fused to the Am targeting peptide (Fig. 2 9 ). GFP fusions to native full length tomato GTK behaved aberrantly possib ly due to misfolding as a result of the appended GFP moiety, so GFP was replaced by the much smaller M yc tag. The tagged GTK protein without its targeting peptide localized only in the cytosol, whereas the native full length construct localized mainly to m itochondria and partly to plastids (Fig. 2 10 ). Collectively, these in vitro and in vivo Am and GTK have alternative start sites, (ii) that if the second site is used the resulting protein remains in the cytosol, and (iii) th at if the first site is used the protein is sent to plastids Am and mitochondria and plastids in the case of GTK. Growth and Metabolic P henotypes of B. subtilis Am D isruptants B. subtilis Am and GTK genes are adjacent and clustered with Met salvage genes ( Fig. 2 2C), and in which Met salvage has been well studied (Sekowska and Danchin, 2002) We therefore used B. subtilis as a model to invest Am to Met salvage by ablating each gene and evaluating the effect on growth on ED minimal medium with 5

PAGE 37

37 methylthioribose as sole sulfur source. On this medium, the sulfur nutrition of the cell depends entirely on the Met salvage cycle ( Fig. 2 1A). Gene ablation was achieved by insertional mutagenesis using the pJM103 plasmid. Unlike that used previously in similar experiments (Sekowska and Danchin, 2002) this plasmid has no promoter and so cannot generate confounding art ifacts by adventitious activation of downstream genes. On 5 methylthioribose medium, the GTK disruptant showed no growth defect Am strain showed a slight lag that was evide nt at 19 h but not 42 h ( Fig. 2 11 ). None of the strains grew in the absence of 5 methylthioribose as sulfur source, and neither disruptant strain manifested any growth defect when sulfate replaced 5 methylthioribose (not shown). The lack of effect of inactivating GTK agrees with the earlier study (Sekowska and Danchin, 2002) Am disruptant is unlike the previously reported heterogeneous pattern in which a few cells formed colonies while most did not (Sekowska and Danchin, 2002) This more complex phenotype may have been due to artifactual gene activation, as noted above. Am is essential for the function of the Met salvage cycle in B. subtilis they leave open the possibility that both enzymes normally carry a significant flux that, in disruptants, is rechanneled through other aminotransferases. As a driver for such rechanneling could be an expansion of the KMTB pool, we attempted to measure the size of this pool in wild type and G TK disruptant cells grown in liquid ED medium with 5 methyl thioribose as sole sulfur source. The KMTB contents of both strains were, however, below the detection limit of

PAGE 38

38 the highly sensitive fluorometric HPLC method used (Pailla et al., 2000) This detect ion limit was 0.4 nmol mg 1 protein which corresponds to an intracellular concentration of ~0.1 mM (Poolman et al., 1987; Burgess et al., 2006) Discussion Am function in bacteria. First, they make a strong a priori case that these enzymes work in tandem to close the Met salvage cycle in B. subtilis and other bacteria. The characteristics of the B. subtilis enzymes reinforce this case; previous work had suggested no concrete role in Met salvage for either enzyme (Berger et al., 2003; Am could not be confirmed by a gene knockout a pproach is perhaps unsurprising, given that other B. subtilis aminotransferases can mediate KMTB transamination with various amino acids, at least if the KMTB concentration is sufficiently high (Berger et al., 2003). Second, the comparative genomic data in Am commonly work together in Am co occur, often as neighbors, in many bacterial genomes that lack genes for Met salvage. One such process, proposed for mammals (Cooper, 2004), c ould be to prevent accumulation of keto acids formed by side reactions of aminotransferases with amino acids that are not their primary substrates. The modest activities of the B. subtilis and plant GTKs with phenylpyruvate an ketoisocaproate are congruent with Am system equips it to dispatch the keto acids back to the parent amino acids. The third genomics based Am frequently occurs Am must have a function that does not depend on GTK. One possibility known from bacteria

PAGE 39

39 ketosuccinamate, the transamination product of asparagine. The in vitro characteristics of maize and to Am show that, like their B. subtilis counterparts, they could potentially close the Met salvage cycle, and their co expression with each other and with 5 methylthioribose kinase in Arabidopsis is consistent with this scenario. If this scenar Am would be expected to be cytosolic proteins since this appears to be the case for the other plant Met salvage enzymes (Sauter et al., 2004; Sauter et al., 2005). The presence of predicted N terminal targeting sequences on both GT Am thus, at first sight, seemed to weigh against a function in Met salvage. Our experimental data, however, provided clear evidence that both proteins have a conserved alternative start site that eliminates the targeting peptide, that this site is used in vivo, and that a fraction probably large Am proteins is consequently localized in the cytosol. Such alternative start sites have various precedents in plants and have been seen as an evolutionary solution to increase the number of cellular functions without multiplying genes (Wamboldt et al., 2009; Yogev and Pines, 2011). Besides the cytosol, the in vivo Am is sent to plastids alone whereas GTK goes to both plastids and mitochondria. The in vitro eviden ce suggests the same, for both proteins were imported into chloroplasts but only GTK was imported into mitochondria. That mitochondrial GTK import was not accompanied by proteolytic cleavage may be because the cleavage site was compromised by the Met to le ucine change that the experiment demanded. If GTK alone enters mitochondria, it would appear to constitute Am always work together. However, as plants

PAGE 40

40 Am like proteins that have not been tested for act ivity against KGM (e.g. Piotrowski et al., 2002) and could be organellar, it is possible that GTK works with one of these. amidase activity has been long known in plants in relation to the hydrolysis of the a sparagine ke tosuccinamate and its hydroxysuccinamate (Lloyd and Joy 1978; Streeter, 1977; Zhang et Am proteins had low activity against these substrates. These enzymes may thus function independently of GTK in pla nt asparagine metabolism. Consistent with this notion, it was recently shown that the Am has high activity against hydroxysuccinamate, and that its ablation causes accumula tion of the two latter compounds (Zhang and Marsolais, 2014). Interestingly, in ketosuccinamate (Zhang and Marsolais, 2014) whereas the tomato and maize enzymes respectively showed 11 and 9 fold preferences for KGM. These striking species differences in substrate preference suggest that KGM deamination is a general function ketosuccinamate deamination is a more taxon specific one. Experiment al Procedures Bioinformatics Protein seq uences were taken from GenBank, MaizeSeq u ence.org, Solgenomics.net, and the SEED database (Overbeek et al., 2005) Comparative genomics analyses of bacterial genomes were made using SEED tools (Overbeek et al., 2005) Sequence s were aligned with Multalin (Corpet, 1988). Organellar targeting

PAGE 41

41 was predicted using TargetP (Emanuelsson et al., 2007) and Predotar (Small et al., 2004). Arabidopsis gene co expression was analyzed using ATTED II tools (Obayashi et al., 2011). Chemicals Chemicals and enzymes were from Sigma Aldrich, unless stated otherwise. KGM ketosuccinamate were prepar ed and quantified as described (Jaisson et al., 2009) Methylthioribose was prepared by hydrolysis of 5' methylthioadenosine in 0.05 M H 2 SO 4 at 100 C for 3 h. The hydrolysate, which contains adenine and 5 methylthioribose, was run through a 1 ml Dowex 50 column (H + form) to remove adenine an d remaining traces of the starting material After removal of SO 4 2 by adding Ba(OH) 2 and centrifugi ng, the solution was conce ntrated under reduced pressure (Schlenk et al., 1973) Am H omologs Maize ( Zea mays ) cDNAs corresponding to GTK homolog GRMZM2G067265 (ZM_BFb0181K01 and Z Am homolog s GRMZM2G169365 and GRMZM2G156486 (Zm_BFb0147P08 and Zm_BFb0221G16, respectively) were obtained from the Arizona Genomics Institute (Tucson, AZ, USA) and sequenced. Both Am cDNAs encoded full length proteins; the two GTK sequences were incomplete and were spliced together using Sequence Overlap Extension ( Heckman and Pease, 2007) to produce a full length sequence. Tomato ( Solanum lycopersicum cv. Floridade ) cDNA provided by H.J. Klee (University of Florida) served as template to amplify the sequenc es of GTK homolog Solyc11g013170 Am homolog Solyc08g062190 Amplifications used PfuTurbo DNA polymerase (Stratagene) and primers ( Fig. 2 12 ) designed with restrict ion sites to allow cloning of amplicons into pET28b (Novagen) for

PAGE 42

42 A m proteins encoding a C terminal His tag, or into pET43.1a (Novagen) for overexpression of GTK proteins containing an N terminal Nus tag to improve solubility. All sequences were truncated to remove putative ta rgeting peptides ( Fig. 2 7 ). Sequence verified constructs were introduced into Escherichia coli strain BL21 (DE3) carrying rare codon plasmids RIPL (Stratagene). Transformants were D 1 thiogalactopyranoside (IPTG) at an OD 600 of 0.6 and grown for a further 4 h at room temperature, except in the case of B. subtilis Am which was induced at 37C. His tagged proteins were purified using Ni NTA resin (Qi agen) according to the manufact phosphate was used in place of sodiu m Am proteins. Purified proteins were desalted on PD 10 columns (GE Healthcare) equilibrated with 50 mM potassium Am pH 8.0), 30 mM KCl mercaptoethanol, and 10% (v/v) glycerol, frozen in aliquots in liquid n itrogen, and stored at 80C. Nus tagged tomato and maize GTKs were purified by Ni affinity chromatography as above, followed by a size exclusion step to remove breakdown products. A Superdex 200 HR 10/30 column was eluted (0.4 ml/min) with 50 mM Hepes, pH 7.4, containing 30 mM potassium chloride (KCl) mercaptoethanol and 5% (v/v) glycerol. Proteins were quantified usin g the Bradford method (Bradford, 1976) with bovine serum albumin as a standard. Size exclusion chromatography Am proteins use d a Superdex 200 HR 10/30 column eluted (0.4 ml/min) with 100 mM potassium phosphate, pH 8.0, containing 0.1 M KCl. Enzyme A ssays contained 50 mM Tris mercaptoethanol, and the specified concentrations of KGM. Incubation was for up to

PAGE 43

43 1.5, at 37C for B. subtilis Am and 25C for the plant enzyme s. The enzyme activities used were low enough to prevent the rate of the KGM r ing opening reaction ( Fig. 2 1B) becoming limiting, the open chain for Am ( See Appendix B ) (Cooper, 2004) Am kinetic studies a 2,4 dinitrophenylhydrazine procedure adapted to a 96 well plate format, ketoglutarate formation (Cooper et al., 1985; Krasnikov et al., 2009) A coupled assay with glutamate dehydrogenase was used to determine enzymatic activity against additional substrates (Jaisson et al., 2009) ntained 100 mM potassium phosphate, pH 7.5, 50 M pyridoxal 5' phosphate, and the specified concentrations of KMTB and L glutamine. Incubation was at 37C for B. subtilis GTK and 25C for plant enzymes; assays were run for up to 1 h. Met formation was dete rmined by HPLC after derivatization with o phthaldialdehyde (Ravanel et al., 1995). An appropriate dilution of the assay was made in 100 mM potassium phosphate, pH 7.5, 10 l of which was derivatized with 90 l of a solution containing 27 mg o phthaldialde hyde in 0.5 ml absolute ethanol, 4.5 ml 400 mM b oric acid NaOH, pH 9.5, and 2% (v/v) mercaptoethanol. After mixing i n the sample loop, derivatized samples were injected onto a Spherisorb ODS 2 RP 18 column (250 x 4.6 mm; 5 m particle size). Buffers use d were: A, 85 mM sodium acetate and 6% (v/v) acetonitrile (pH 4.5); B, 60% (v/v) acetonitrile. Linear gradients were: 40 to 80% B, 0 to 6 min; 80% B, 6 to 9 min; 80 to 40% B, 9 to 10 min; 40% B, 10 to 12 min (40C, 1ml/min). o phthaldialdehyde derivatives were measured fluorometrically (340 nm excitation, 455 nm emission).

PAGE 44

44 Bacillus subtilis E xperiments Bacillus su btilis subsp. subtilis str. 168 knockout strains were constructed using B. subtilis (Perego, 1993). Nucleotides 30 359 of the B. subtilis GTK gene ( mtnV ) and nucleotides 41 329 of the B. subtilis Am gene ( mtnU ) were PCR amplified and cloned into the BamHI site of pJM103. The sequence verified c onstructs were transformed into B. subtili s u sing the two step procedure (Cutting and Vander Horn, 1990); gene knockout was verified by PCR using primers listed in Fig. 2 12 B. subtilis was cultured in ED minimal medium (Sekowska and Danchin, 2002) consisting of: K 2 HPO 4 8 mM; KH 2 PO 4 4.4 mM; glu cose, 27 mM; Na 3 citrate, 0.3 mM; L glutamine, 15 mM; L 4 2 mM; MgCl 2 0.61 mM; CaCl 2 3 2 2 2 2.52 2 2 MoO 4 used as sulfur source (0.1 mM), MgSO 4 was replaced by MgCl 2 at the same magnesium concentration (2 mM). For growth on plates agarose (0.8%) was used as solidifying agent. Cultur es (50 ml) for KMTB analysis were harvested at early stationary phase and fro zen in liquid nitrogen. Cell pellets were resuspended in 1 ml of cold 0.1 N HCl and sonicated on ice th ree times for 10 s. The lysate was incubated on ice for 30 min, cleared by cen trifugation at 4C for 5 min at 15 000 x g and evaporated to d ryness (Ishe rwood and Niavis, 1956; Siegel et al., 1977). K et o acids were d erivatized with o phenylenediam ine (OPD) as described (Pailla et al., 2000) except that the derivatization Ketovalerate was used as internal standard. Separation and fluorescence detection were as described (Pailla et al., 2000)

PAGE 45

45 Dual Import Assays Full length tomato and maize cDNAs were clo ned into pGEM 4Z (Promega) using forward pri mers containing a Kozak sequence; cloning sites are given in Fig. 2 12 To ablate putative second start sites, A TG codons were changed to TTG (l eu cine ) by using entire constructs as templates in PCR reactions that included the mutation carrying primers. Sequence verified constructs were introduced into E. coli TOP10 cells (I nvitrogen). C oupled in vitro transcription /trans lation, organelle isol ation dual import assays, and positive control proteins for organellar import were as described (Rudhe et al., 2002; Zallot et al., 2013). Import reactions were run for 10 min for GTK and Am, 5 min for the chloroplast positive control (At1g60990), and 20 min for the mitochondrial positive control (soybean alternative oxidase). Transient Expression and Microscopic Analysis of GFP F usion Pr oteins in BY 2 C ells Full length or N terminally truncated GTK cDNAs were cloned into the Xba I site which is immediately upstream of the Myc epitope sequence of pRTL2/ Nhe I myc (Clark et al., 2009). Full length or N Am cDNAs, or their predicted targeting peptide, were cloned into the NcoI site of pUC18/ Nco I mGFP (using BspH I if an internal Nco I site was present). The vector pUC18/ Nco I mGFP, has a C a MV 35S promoter and a unique Nco mon omeric green fluorescent protein (GFP) (Clark et al., 2009). Primers used for each construct are given in Fig. 2 12 Targeting peptides were predicted from alignments of the N Am homolog ues. The targeting regions that were removed, or fused directly to GFP, are shown in Fig. 2 7

PAGE 46

46 Tobacco (Nicotiana tabacum cv. Bright Yellow 2 [BY 2]) suspension cell cultures were maintained and prepared for bombardment as described (Lingard et al., 2008). Transient (co )transformations were performed using 4 g plasmid DNA precipitated onto tungsten or gold microcarriers (Bio Rad), along with a Biolistic PDS 1000/HE particle delivery system (Bio Rad). Bombarded cells were incubated for ~8 h to allow expre ssion and sorting of the introduced gene products. Cells were fixed in 4% (w/v) formaldehyde, then permeabilized with 0.01% (w/v) pectolyase Y 23 (Kyowa Chemical Products) and 0.3% (v/v) Triton X 100 (Sigma cytochrome c oxidase subunit I I (CoxII) anti bodies and goat anti rabbit Rhodamine Red X IgGs were as N acetyl glutamate kinase (NAGK) antibodies have been also described (Chen et al., 2006). Epifluorescent images were acquired using an Axiosco pe 2 MOT epifluorescence microscope (Carl Zeiss) with a 63 Plan Apochromat oil immersion objective. Images were captured using a Retiga 1300 CCD camera (Qimaging) and Openlab software (Improvision); figures were composed using Adobe Photoshop CS (Adobe Sy stems). Micrographs shown in the figures are representative images obtained from viewing >50 cells from at least two replicate biolistic experiments.

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47 Table 2 1 K inetic constants of recombinant GTK ketomethylthiobutyrate. Activities were determined by measuring formation of Met as its o phthald ialdehyde derivative by HPLC The concentration of glutamine was 20 mM. Values are means of three replicates SE. GTK K m (mM) K i (mM) k cat (s 1 ) k cat / K m ( m M 1 s 1 ) Tomato 0.22 0.011 0.38 0.03 0.15 0.004 0.71 0.02 Maize 0.07 0.003 1.61 0.04 0.12 0.002 1.73 0.04 B. subtilis 1.91 0.5 19 3.73 1.58 2.32 0. 467 1.26 0.09

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48 Table 2 2 Activities of recombinant amidase amide substrates. Substrate concentrations were 15 mM. Activities were determined by enzymatic assay of NH 3 release. Maize 1, GRMZM2G169365; Maize 2, GRMZM2G156486 Values are means of three replicates SE. Substrate Amidase activity (nkatal mg 1 protein) Tomato Maize 1 Maize 2 Ketoglutaramate 1180 17 475 28 537 15 Ketosuccinamate 106 4 35 5 36 6 S uccinamate 745 15 179 13 102 15 Glutamine <1 <1 <1 Asparagine <1 <1 <1 Butyramide <1 <1 <1

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49 Table 2 3 K inetic constants of recombinant amidase ketoglutaramate as ketoglutarate as its 2,4 dinitrophenylhydrazone. Maize 1, GRMZM2G169365; Maize 2, GRMZM2G156486. Values are means of three replicates SE. Values for kc at and Km for B. subtilis Am were not calculated as the protein could not be purified in active form. Amidase K m (mM) k cat (s 1 ) k cat / K m ( m M 1 s 1 ) Tomato 6.49 0.95 72.7 10.4 11.32 0.80 Maize 1 6.42 0.73 63.1 7.1 9.87 0.29 Maize 2 5.31 0.79 50.8 3.3 9.98 0.97

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50 Figure 2 1. The methionine salvage pathway. ( A ) Steps in the Met salvage pathways in bacteria (gene names in italics) and plants (gene names underlined). Alternative bacterial steps are shown as gray arrows. Abbreviations: MTA, methylthioadenosine; MTR, methylthioribose; MTR 1 P, 5 methylthioribose 1 phosphate; MTRu 1 P, methylthioribulose 1 phosphate; DKP 1 P, 2,3 diketo 5 methylthiopentyl 1 phosphate; HKMP 1 P, 2 hydroxy 3 keto 5 methylthiopentenyl 1 phospha te; DHKMP, 1,2 dihydroxy 3 keto 5 methylthiopent ketomethylthiobutyr ate. ( B ) The tandem transamination and deamination reactions mediated by glutamine transaminase K (GTK) and amidase ( Am) that are thought to close the salvage cycle in mammals. ketoglutaramate spontaneously cyclizes to 5 hydroxy 2 oxoproline and that at neutral pH the equilibrium favors the ring form (99.7%) over the open chain fo rm (0.3%) (Cooper, 2004).

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51 Figure 2 2. amidase homologs. ( A ) Distribution among major bacterial taxa of genes for amidase ( Am), and of Met salvage genes mtnA and mtnD Major taxa are represented by one or a few species. Genes that are clustered on the chromosome are connected by bars. Comparative analyses of 588 representative bacterial genomes were made using the SEED and its tools; full results ar subsystem at http://pubseed.theseed.org/ ( B ) Bar chart and Venn diagram summarizing of the distribution and clustering of GTK, Am, mtnA mtnD in bacterial genomes. ( C ) Example s of clustering of bacterial GTK and Am genes with each other and with genes for Met salvage (in green) or Met transport (in yellow). Genes in gray have functions unrelated to Met. Arrows point in the direction of transcription. Note that the examples co me from diverse taxa and that the adjacent GTK and Am genes can be in either order on the same or opposite DNA strand. (D) ATTED custom co expressed gene network around Arabidopsis Am (At5g12040) and GTK (At1g77670) genes. O ther network genes include t he Met salvage gene 5 methylthioribose kinase (MTK, A t1g49820), and the glutamate or aspartate related genes IDH (isocitrate dehydrogenase, At5g14590), OTC (ornithine transcarbamylase, At1g75330), QPRT (quinolinate phosphoribosyltransferase, At2g01350), and ADSS (adenylosuccinate synthetase, At3g57610).

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52

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53 Figure 2 3 Purification of recombinant bacterial and plant Am and GTK proteins. (A) Ni affinity purified recombinant GTK proteins: 1 B. subtilis ; 2 Tomato (N terminal Nus tag); 3 Maize (N terminal Nus tag). Nus tagged proteins underwent an additional size exclusion step to remove breakdown products. (B) Ni affinity purified recombinant Am proteins: 1 Tomato; 2 GRMZM2G169365; 3 GRMZM2G156486; 4 Empty pET28b soluble, desalted fr action; B. subtilis soluble desalted fraction.

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54 Figure 2 4 Analytical size exclusion chromatography of recombinant plant Am and GTK proteins. Tomato, and two maize (GRMZM2G169365, GRMZM2G156486) Am proteins with calculated molecular masses of the dimers as 55, 54, and 52 kDa, respectively. Nus tagged maize and tomato GTK proteins as well as B. subtilis GTK were also found as dimers with calculated molecular masses of 432, 410, and 108 kDa, respec tively. Open circles are calibration standards (kDa): cytochrome c (12.4), carbonic anhydrase (29), albumin (66), alcohol dehydrogenase (150), b amylase (200), apoferritin (443), and thyroglobulin (669).

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55 Figure 2 5 Substrate preferences of plant and bacterial GTK proteins. Purified recombinant GTKs from tomato, maize, and B. subtilis were assayed with various amino donors and acceptors. Activities were assayed by measuring formation of amino acid products as their o phthaldialdehyde derivatives by HPL C. Values are expressed relative to the maximal activity observed (1.0) and are means of three replicates SE. The absolute values (katal mg 1 protein) for the tomato, maize, and B. subtilis enzymes were: 0.01, 0.45, 9.88 and 0.01, 0.27, and 13.77 for the amino donor and amino acceptor experiments, respectively. The threshold for activity detection for all enzymes was 0.3% of that with KMTB and glutamine as substrates. (A) Amino donor preferences. KMTB (5 mM) was used as amino acceptor. Amino acids used as amino donors are identified by their single letter codes. (B) Amino acceptor keto acids used as amino acceptors were: KMBT, phenylpyruvate (PhPyr), keto isovalerate (K keto methylvalerate (KMV).

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56 Figure 2 6 Substrate inhibition of recombinant GTK enzymes. Non linear regression curve fitting to the equation in A was used to estimate kinetic parameters for maize (B), tomato (C) and B. subtilis (D) GTK enzym es. Values are means of three replicate determinations SE. See text for kinetic parameters.

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57 Figure 2 7 N Am proteins. Sequences were obtained from EST and genome databases. Putati ve alternative start Met residues are highlighted green. Red arrows indicate the first strongly conserved residue, inferred to be at the beginning of the mature protein. For tomato and maize proteins, the putative targeting sequence was removed for recombi nant protein production and for subcellular localization studies (GFP and dual import). The adjusted start locations are underlined and bolded (these locations are the alternative start Met in Am proteins and a Met inserted immediately before the indicat ed residue in GTKs). Representative mammalian (human) and bacterial ( Bacillus subtilis Nocardia Clostridium ) sequences are included for comparison.

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58 Figure 2 8. In vitro Am and GTK. (A) Full length cDNAs (FL), full length cDNAs with the putative alternative start Met Am, Met65Leu; for GTK, Met43Leu), and truncated cDNAs beginning at the putative Am, 42) were cloned into pGEM 4Z. Constructs acted as template for in vitro transcription and translation by a wheat germ system in the presence of [3H]leucine. Translated proteins were resolved by SDS PAG E and visualized by fluorography. The positions of molecular weight markers (kDa) are indicated. (B) Full length cDNAs with the alternative start Am and GTK M2L), plus controls for mitochondrial (soybean alternative oxidase, GmAOX ) or chloroplast (At1g60990) import, were transcribed and translated in vitro using a rabbit reticulocyte system plus [3H]leucine. The translation products were incubated in the light with mixed chloroplasts (C) and mitochondria (M), which were then repuri fied on a Percoll gradient, without ( ) or with (+) prior thermolysin treatment to remove non imported proteins. Note the faint but definite signal in the GTK M2L lane containing thermolysin treated mitochondria, and the absence of such a signal for the co ntrol plastidial protein At1g60990, which has a stronger chloroplast import signal. Proteins truncated at the alternative start Met were, as expected, not imported into chloroplasts (not shown). Proteins were separated by SDS PAGE and visualized by fluorog raphy; exposure times were adjusted to give comparable band intensities. Samples were loaded on the basis of equal chlorophyll or mitochondrial protein content next to an aliquot of the translation pro duct (P). The positions of molecular weight markers (k Da) are indicated.

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59

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60 Figure 2 9 Representative confocal images of tobacco BY 2 cells transiently Am constructs C terminally fused to GFP. Cells were 64 Am G FP), wild type full Am GFP), or predicted plastid N terminal targeting sequence only (1 Am GFP), fused to GFP. After ~8 h, cells were formaldehyde fixed, immunostained for endogenous N acetyl glutamate kinase (NAGK), serving as a plastid mar ker protein, and examined by confocal microscopy. Each row of images corresponds to the fluorescence attributable to the candidate fusion protein and NAGK immunostaining (green or red, respectively), and the corresponding merged image. Solid arrowheads ind icate examples of colocalization of the expressed protein and endogenous NAGK in plastids. No mitochondrial targeting was observed when cells were immunostained for endogenous mitochondrial cytochrome c oxidase subunit II (not shown). Bar in top left panel equals 10 m.

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61 Fig ure 2 10 Representative epifluorescence images of tobacco BY 2 cells transiently expressing tomato GTK constructs with a C terminal appended Myc tag. Cells were biolistically bombarded with plasmid DNA encoding either a truncated 42 GTK Myc) or native full length (GTK Myc) Myc epitope tagged protein. After ~8h, cells were formaldehyde fixed, immunostained for the endogenous cytochrome c oxidase subunit II (CoxII), serving as mitochondrial marker protein, or plastidial NAGK, and ex amined by epifluorescence microscopy. Each row of images corresponds to the fluorescence attributable to the candidate fusion protein and CoxII or NAGK immunostaining (green or red, respectively), and the corresponding merged image. Hatched boxes represent the portion of the cell shown at higher magnification in the panels below. Solid arrowheads present in high magnification images indicate examples of colocalization of the expressed protein and endogenous NAGK in plastids. Truncated GTK Myc was found not to colocalize with anti NAGK immunostained cells (data not shown). Bar in top left panel equals 10 m.

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62 Figure 2 11 Growth of B. subtilis with 5 methylthioribose as sole sulfur source. Three independent isolates of wild type (WT), Am ( mtnU ), and GTK ( mtnV ) strains were grown at 37C for 19 or 42 h on ED minimal medium containing 27 mM glucose, 15 mM L glutamine, 0.1 mM 5 methylthioribose as sole sulfur source, and 0.8% agarose.

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63 Figure 2 12. Synthetic oligonucleotides used in this study.

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64 CHAP TER 3 GLUTAMINE TRANSAMINASE K AND OMEGA AMIDASE ANTISENSE LINES IN TOMATO Background The Impact of Met and AdoMet on Growth, Development and Genome Stability Methyltransferases rely upon AdoMet as universal methyl donor and have a vast array of biological targets In plants and animals, the organization of chromatin structure is largely affected by the methylation patterns of histone proteins and DNA ( He G et al., 2011; Roje, 2006). These methylation patterns contribute to the epigenetic landscape of the eukaryotic genome and are involved in diverse biological processes (He G et al., 2011; He X et al., 2011). With respect to the plant genome, DNA methylation participates in the suppression of the activity of transposons and other r epetit ive sequences, as well as regulating gene expression (He G et al., 2011; He X et al., 2011) Some of these epigenetic marks are transferred through to the next generation in a process known as imprinting ( Gehring 2013). Histone modifications occur as post translational covalent modifications at the N terminal tail of the histone protein (He G et al., 2011). The se modification s are also important to the regulation of gene expression and response to environmental inputs including stress, pathogen attac k, temperature, and light ( Pfluger and Wagner 2007) In addition to the role of AdoMet as a methyl donor i n plant growth, development, and genome stability, its role as a precursor for ethylene and polyamines adds to the biological importance of this co mpound. Ethylene is also known to be involved in regulation of growth, development, and responses to stress and pathogen attack in plants (Bleecker and Kende, 2000). P olyamines (PAs) are found in all living organisms play roles in development and stress responses and are positively charged at cellular

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65 pH values PAs are also known to chemically interact with DNA, RNA, phospholipids, and some proteins (Roje, 2006; Sauter et al., 2013). With AdoMet underpinning such important biological processes we were interested in the role that the Met salvage cycle has in planta Particularly during green fruit maturation the process of endoreduplication is occurring ( Bourdon et al., 2012) and with this increase in DNA synthesis, the metabolic load of polyamin e biosynthesis also i ncrease s ( Paschalidis and Roubelakis Angelakis 2005 ). During r ipening there is an increase in ethylene biosynthesis along with an increase in transcription of the E8 fruit specific promoter ( Deikman et al., 1992). These biological phe nomena are the main reasons why tomato was selected as the model system for determining in planta Am antisense lines. Tomato as a Model System The major advantage of us ing tomato as the model system as ide from its genetic and genom i c resources, is the fact that modifying a specific tissue (i.e. fruit) limits potential deleterious effects for the whole plant. Considering the mild growth phenotype observed for the B. subtilis Am, and no observable growth phenotype for B. subtilis GTK (as described in Chapter 2) we hypothesized that the primary validation of gene function would be perturbation of the KMTB and KGM pools. The fruit is an ideal system for evaluating such effects because fruit tissues undergo periods of rapid cell div ision and expansion. During these times, there are str ong require ments for Met and AdoMet associated meta bolism. This will allow for any direct effects on metabolite pools to be readily assessed in fruits over short or extended time periods.

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66 Design of Ant isense Constructs In order to limit the transgene expression to the fruit, we used two promoters. The first was the E8 promoter, which is active only in fruit tissues, is ripening specific, and ethylene responsive (Deikman et al., 1992; Deikman et al., 199 8). The second was the Tfm7 promoter, which is expressed in all fruit tissues from anthesis to the onset of ripening ( Santino et al., 1997) These two promoters were selected with the goal of limiting the expression Am when there was a large metabolic burden on the Met salvage cycle For the E8 promoter the metabolic load would be ethylene biosynthesis and for the Tfm7 promoter it would be polyamine biosynthesis, during endoreduplication as the green fruit matures. A constitutive promoter, t he Figwort Mosaic Virus (FMV) promoter was also used to determine if there would be a metabolic perturbation if the expression of Am was universally limited. Experimental Plan for Transgenic Tomato Lines The approach used to produce antisense l ines is to first obtain at least 20 i ndependent transgenic events for each construct. This is followed by the use of quantitative RT PCR to choose, for each construct, three or more independent events that exhibit maximal reduction in gene expression. The Micro Tom cultivar was selected to efficiently use the growth space that was available. The overall goal after 3 or more lines with maximal reduction in gene expression, were selected, was to evaluate both metabolite profiles and overall phenotypes in T 1 generation plants For E8 antisense lines, time from breaker stage to red ripe was to be measured, in addition to ethylene synthesis. For TFM7 antisense lines, time from anthesis to breaker stage (onset of ripening) was to be measured, along with fruit size, and ethylene synthesis. Overall growth effects in the FMV antisense lines was to be

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67 assessed by measuring seedling growth rates, time to first flowering, and time from anthesis to fruit ripening. Ethylene synthesis, which as stated above, depends o n AdoMet, was to be assessed during fruit ripening. These lines were to also be grown for metabolomic profiling, with the fruit as the target tissues for plants containing the E8 and Tfm7 constructs. Target tissues for plants containing the constitutive FMV construct would be roots, stem, leaf and fruit. KMTB is commercially available as a standard, and KGM as a standard will be prepared as described in Chapter 2. Results Expression Analysis of Antisense Tomato Lines Tomato lines were confirmed to contain the antisense expression construct before RNA was extracted from either fruit or leaves of t he transgenic plants. WT Micro Tom seeds were germinated for parallel growth u pon arrival of the transformed plantlets, which were prepared at the University of Nebraska Expression analysis by quantitative PCR (qPCR) for all lines transformed with the three antisense GTK constructs displayed no reduction in expression levels relative to GTK expression in WT fruit or leaves (Figure 3 1). The two mo st likely explanations for these results are (i) t he annealing site of the primers overlapped with the sequence that would have been expressed strongly under each respective promoter (ii) t he construct was not at all effective at producing an antisen se mRN A which would then have resulted in reduction of measurable transcript. To address the first possibility, the second primer set that was untranslated region of the tomato GTK of the antisense construct and thus this primer pair should have been able to determine if the primer design was the issue at hand. As presented in Figure 3 1 each primer set

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68 gave similar results, where th ere was no reduction in transcript abundance of GTK. This suggests that the second explanation, where the construct was ineffective is more likely to be the cause of this result. Am constructs, at least one line of e ach set of Am levels (Figure 3 2). In the case of pMON10086 Am 2 had just under 10% expression (Figure 3 2 A). Transcript knockdown for pHK Tfm7 Am was not as dramatic as about 30% transcript level remained detectable (Figure 3 2 B). The construct encoding the constitutive FMV promoter resulted in one line with <10% Am 10), and several other lines with approximately 20% A m (Figure 3 2 C). While there were still transgenic plant lines where transcript level was relatively unchanged compared to WT, the fact that some lines had a drastic reduction con firmed that the antisense construct design (same strategy for each gene) wa s effective for at least one of the genes of interest. Discussion Considering that no growth phenotype was observed for the GTK B. subtilis mutant work described in Chapter 2, it seems reasonable to extrapolate the biological possibility that other transam inases would act on KMTB in tomato as well. Perhaps even if GTK were to carry the majority of the flux in vivo for the Met salvage cycle, upon knockdown of GTK, KMTB would accumulate to the point where other transaminases would potentially act on this sub strate. It seems plausible that high resolution metabolomics analysis would have been able to detect such a (potentially minor) Am antisense lines that exhibited the most severe reduction in measurable transcript would be well suited to determine if there was in fact

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69 a metabolic phenotype, where the concentration of KGM would be elevated. Upon successful determination of a metabolic phenotype through targeted metabolomics, it would have been interesting to analyze if metabolic perturbations rippled through the rest of methyl group metabolism. If significant perturbations in methyl metabolism were identified, targeting bisulfite sequencing could be an appropriate follow up experiment Considering the tomato methylome has recent ly been published ( Zhong et al., 2013) genomic loci affecting growth and development could be probed to determine if these perturbations persisted through to epigenetic changes. As metabolite repair enzymes, the activity of GTK and Am would be integral to normal growth and development. During times of stress, the failure of these enzymes could threaten not just the metabolic pathways discussed above but also the epigenome. Perhaps, based on B. subtilis Am knockdow n plants provided the best opportunity to determine the in planta metabolic Am knockdown lines would be worthwhile to pursue for the purpose of determin ing whether a metabolic phenotype exists. Experimental Procedure s Bioinformatics Protein seq uences were taken from GenBank and Solgenomics.net Antisense untranslated region with the goal of added siRNA specificity. Primers used for expression analysis were designed using Primer Express recommendations (optimal melting point, 60C; optimal primer length, 20 bp; amplicon length, 60 80 bp). These and primers used for cloning are listed in Figure 3 3.

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70 Cloning To mato ( Solanum lycopersicum cv. Floridade ) cDNA provided by H.J. Klee (University of Florida) served as template to amplify the sequences of GTK Solyc11g013170 Am Solyc08g062190 used for preparation of antisense constructs Amplifications used PfuTurb o DNA polymerase (Stratagene) and primers (Fig. 3 3 ) designed with restrict ion sites to allow cloning of amplicons into pMON10086, pGen7nos, and pFMV. In the case of the latter two constructs, NotI was used to excise and subclone the promoter and antisens e sequence into the binary vector pHK1001.All constructs were confirmed by sequencing and were kindly provided by H.J. Klee (University of Florida). Transgenic Plants Micro Tom tomato seeds ( Ball Seed Company ) were sent along with sequenced constructs to t he Plant Transformation Research Core Facility at the University of Nebraska for transformation with Agrobacterium strain ABI Plantlets containing the pMON10086 GTK, pHK Tfm7 Tfm7 GTK, pHK FMV FMV GTK construc t were i dentified by PCR performed on genomic DNA prepa red using primers located within the Kanamycin resistance gene in each construct and primers located within the naturally occurring ACC gene were used as a genomic DNA control. Plants were grown at 23 C for 16 h of light. Plants transformed with pMON10086 and pHK TFM7 had their fruit harvested, seeds removed and the fruit tissue was frozen in liquid nitrogen and stored at 80 C until RNA was extracted. Leaf samples were removed from plants transformed with pHK FMV frozen in liquid nitrogen

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71 and stored at 80 C until RNA was extracted. These plants too had seeds removed and stored for future work. Quantitative PCR Total RNA was prepared from fruit tissues using Plant/Fungi Total RNA Purification Kit ( Norgen B iotek ). Possible genomic DNA contamination was removed by DNase treatment (Qiagen). RNA was quantified spectrophotometrically, and 1 g of RNA of each plant line was used for cDNA synthesis. cDNA was synthesized using SuperScript III First Strand Synthes is System for RT PCR (Invitrogen). The provided procedure was followed except for the cDNA synthesis step where the reactions were incubated at 50 C for 30 min followed by 55 C for 1 h. Real time PCR quantification was performed in a 96 well plate with St epOnePlus real time PCR system The primers u sed to amplify DNA fragments of Am are listed in Figure 3 3. Primer concentration was optimized (50 900 nM) and the concentrations leading to the lowest threshold cycle values (CT) were used to calcu late amplification efficiency. Standard conditions were used with EvaGreen 2X qPCR MasterMix ROX ( ABM) (95C for 10 min and 40 cycles of 95C for 15 s, 60C for 1 min, and 72C for 30 s). The 2 C T quantification method (Schmittgen and Livak 2008) was used for quantification, with an actin gene (GenBank XM_004235020 ) as internal reference. Gene of interest (GTK or Am) expression levels were expressed relative to levels found in the appropriate WT sample (fruit or leaf cDNA).

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72 Figure 3 1. Transcript level of GTK in transgenic tomato lines. Transgenic tomato lines contained: (A) pMON10086 GTK antisense construct; (B) pHK Tfm7 GTK antisense construct; (C) pHK FMV GTK antisense construct. Relative levels of mRNAs were determined by quantitat ive polymerase chain reaction (qPCR) using the 2 T quantification method with actin as internal reference. Values are means and SD from three technical replicates. Results are expressed relative to GTK levels present in WT fruit (A, B) and WT leaf (C) Two different primer sets were used to corroborate no observed reduction in GTK gene expression.

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73

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74 Figure 3 2. Transcript level of Am in transgenic tomato lines. Transgenic tomato lines contained: (A) pMON10086 Am antisense construct; (B) pHK Tfm7 Am antisense construct; (C) pHK FMV Am antisense construct. Relative levels of mRNAs were determined by quantitative polymerase chain reaction (qPCR) using the 2 T quantification method with actin as internal reference. Values are mean s and SD from three technical replicates. Results are expressed relative to Am levels present in WT fruit (A, B) and WT leaf (C).

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75

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76 Figure 3 3. Synthetic oligonucleotides used in this study.

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77 CHAPTER 4 CONCLUSIONS This study set out to determine whether the evidence that was accumulated through comparative genomic methods could address the gap in knowledge that existed in the Met salvage cycle. The enzymes performing the final step of the salvage cycle had not been definitely assigned. Either there was a specific transaminase that was responsible for this reaction and it had not been identified or several general transaminase s were capable of converting KMTB to Met. Biochemical logic suggests that general transaminases would not be an to the inherent instability of KMTB, and the characteristic of general transaminases to be readily reversible, therefore not minimizing the accumulation of the labile KMTB. This logic, along with comparative ge nomic evidence, both genomic and post genomic led to the hypothesis that a specific type of transaminase, a glutamine dependent transaminase was responsible for the final reaction of this pathway. In m ammalian literature biochemical experiments describ ed glu tamine transaminase K as having a high specificity for KMTB. In addition, the product of this reaction is a unique keto acid KGM, that spontaneously cyclizes at physiological conditions. In the linear form, KGM Am, which hydr transamination step towards Met formation, but they then limit the buildup of the labile KMTB and potentially toxic KGM. Minimizing th e abundance of compounds such as these puts these enzymes in the category of metabolite repair reactions. While biochemical characterization of the tomato, maize and B. subtilis homologs of GTK and Am indicated that these enzymes had activities on the substrates of interest close to

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78 published mammalian and bacterial values, in vivo functional complementation was not successful. Inser Am were prepared in the commonly used gram positive model organism B. subtil i s When the sulfur source in the medium was limited to MTR, an interme diate in the Met salvage cycle there was only a slight growth Am m utants. The GTK mutants had no observable growth phenotype on plates. Attempts at complementin g th e Am mutant were unsuccessful. This left the primary evidence for function of these two enzymes resting on in vitro biochemical experiments. The plant homologs were found to have an N terminal extension in their amino acid seq uence compared to the prokaryotic homologs. This type of extension suggests organellar targeting. When it is considered that the other Met salvage cycle enzymes are all present in the cytosol, a nd this is where Met salvage takes place, organellar targeti ng is a direct contradiction to function if these enzymes are not even present in the required cellular location. Interestingly, it was determined that both the tomato and maize homologs of GTK and Am encoded alternative start sites, which upon experime ntation with fluorescent fusion proteins confirmed that each protein was localized to the cytosol and to plastids (in the case of GTK, mitochondria as well). These results mean that these two enzymes are localized in the same cellular compartment as the p athway in which they are implicated. The presence of alternative start sites, the weak growth phenotypes observed for B. subtilis mutants and t he Am may perform alternate reactions feed into the theme of biolo in vitro Am provide evidence for involvement

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79 in the Met salvage cycle, however, additional biological roles cannot be exclud ed. Selective pressure appears to have retained the genomic proximity and context of Am (Figure 2 2 C) but GTK may not be the sole enzyme capable of acting on KMTB. The stability of KMTB and the toxicity of KGM have been mentioned earlier, but if the fitness or survival of a given or ganism is at stake, it is beneficial to that organism to have multiple means to remove or detoxify these compounds. Natural selection therefore could have been the driving force for alternative enzymes to act on these compounds. The above is just one pot ential explanation for the in vivo B. subtilis results described in Chapter 2. However, it does seem that the comparative genomics evidence and the dual targeting of these enzymes suggest they have a larger biological role. If this is the case for these two enzymes, the biological capacity of other enzymes must be larger than previously thought. Once metabolite repair is a great deal more prevalent than previously imagined.

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80 APPENDIX A ADDITIONAL COMPARATIVE GENOMICS Am to complete the methionine (Met) salvage cycle was presented in Chapter 2. Specifically, comparative genomics techniques provided some of the first evidence f or this functional association. In addition Am were found present, and sometimes clustered, in genomes where the Met salvage cycle was absent (Figure 2 2). This Am may work toge ther in metabolic processes aside from Met salvage. A general role was proposed for mammals (Cooper, 2004) Am could possibly prevent accumulation of abnormal potentially keto acids. Side reactions of aminotransferases with amino acids that are not keto acids. Fitting well with this hypothesis were the modest activities of the B. subtilis and plant GTKs wi th ketoisocaproate. The coupled and irreversible nature of the keto acids to be converted back to the parent amino acids. In an attempt to gain a better understanding of the possible additional metabo lic roles, comparative genomics were once again used to determine if additional hypotheses of function could be established. Associations listed on the STRING database for B. subtilis Am yielded no obvious functional associations that were not a lready tied to Met salvage or Met metabolism. The next line of inquiry was based upon plant co expression data, freely available on ATTED ( http://atted.jp/ ). The underlying reason for using Arabidopsis co expression data is related to the discovery that alternative start sites were found to be responsible for cytosolic and organellar

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81 Am in the cytosol was stated to be with Met salvage, however their role in organelles re mained unclear Therefore, by identifying plant genes that are co Am, any associations found with prokaryotic homologs of these genes may assist with identifying an alternative metabolic (and potentially organellar) role. The Ar Am homologs (At1G77670 and At5G12040, respectively) were used as the input for ATTED. There were three different genes listed in a co expression gene network with GTK or Am (Figure A 1) e ach of which had a strong homolog in B. subti lis The benefit to identifying prokaryotic homologs is that Am to identify any genomic associations. Upon analysis of these genes in the SEED database ( http://pubseed.theseed.org/ ) a rgininosuccinate synthase ( argG ) Am in Blautia hydrogenotrophica While additional clustering of these two genes was not present, of the 4846 genomes that encode A m, 4802 (99%) of those al so encode argG Interestingly additional genes encoding enzymes involved with arginine (Arg) A m, and occasionally GTK as well (Figure A 2), in total 73 genomes were found to have such clustering. Included in these gene clusters was an enzyme encoding an aminotransferase, acetylornithine aminotransferase (ArgD) that converts N acetylglutamate 5 semialdehyde to N acetylornithine (Figure A 2). The co occurrence of A m and argD was again strong where of the 4846 genomes that encode A m, 4323 (89%) also encode arg D. Along

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82 with the clustering evidence, the co occurrence also adds to the evidence that Arg biosynthetic genes may be functionally associated with GTK and A m. The first four steps of Arg bio synthesis are strongly conserved in plants (Slocum, 2005) ; these steps originate with glutamate as outlined in Figure A 2A. The most important detail about the corresponding plant enzym es is that their activity has been found in the chloroplast (Jain et a l., 1987; Slocum, 2005). Specifically, Arabidopsis acetylornithine aminotransferase (At1g80600) was found to localize to the chloroplast ( Frmont et al., 2013). Thus the prokaryotic genomic clustering and this localization information suggest a functional Am and Arg b iosynt hesis. Next experimental steps would be to screen N acetylglutamate 5 semialdehyde or other semialdehydes with similar carbon chain lengths as substrates for GTK In addition, any known damage products of these pathway intermediates would have to be identified and then also screened to determine if any activity can be found for GTK or Am Another potential connection to amino acid metabolism was found when analyzing the genes which clustered around GTK Am. The gene encoding d iaminopimelate epimerase ( DapF, EC 5.1.1.7), which is involved in lysine biosynthesis (Figure A 3) was found to cluster with GTK in Dethiobacter alkaliphilus Upon further analysis it was determined that GTK has close homolo gy to LL DAP aminotransferase (DapL, EC 2.6.1.83) and that the annotation of DapL was not accurate. Once corrected, GTK did not cluster with Lys biosynthesis genes, but of the 8275 genomes that contained d apL clustering with at least one other Lys biosyn thesis gene was found in 37% of those genomes ( 3076 ) (Figure A 3B). In addition to this clustering evidence it is

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83 interesting to note how close in homology GTK proteins are to DapL proteins by means of phylogenetic analysis (Figure A 4). This tree shows that prokaryotic and eukaryotic GTK homologs are found closer to DapL homologs than the other aminotransferases involved in Lys biosynthesis. With this information it would be interesting to determine if there is any overlap in substrate specificity betwe en the two enzymes. It is particularly interesting because in Arabidopsis, there is a DapL homolog that is predicted to be targeted to the chloroplast ( Hudson et al., 2006) If there was an overlap between the substrate specificities of the two enzymes, Am system may be acting upon intermediates in Lys biosynthesis. The first compound of the pathway to assay against GTK would be L 2,3,4,5 tetrahydrodipicolinate as this is the named substrate of DapL. Again furt her inquiry into any common breakdown products of this pathway would provide more substrates to test. These two examples provide a framework from which to determine the organellar Am. While these examples do not suggest a hypothesis as specific as that developed for Met salvage, the y do suggest new com pounds to screen as substrates Chemical knowledge of the compounds in Arg and Lys biosynthesis, as well as being able to predict potential breakdown products would help to fu rther target the next experimental steps. If specific compounds were found to be good substrates in vitro subcellular fractionation could be performed to isolate chloroplasts in an attempt to confirm the activity in vivo T he major issue with this exper iment is that if GTK and Am are in fact performing a reaction that can also be completed by another enzyme(s) there would be no clear way to determine which enzyme was responsible for

PAGE 84

84 the activity. The next step would be to identify t he best bacterial model system in which to generate knockout strains in Arg or Lys biosynthesis pathway These mutants may result in stronger growth phenotypes to be observed to validate in vivo function of GTK and Am in this additional metabolic role.

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85 Figure A Am. ATTED coexpression database listed the above genes as present in a co expression Am. B. subtilis homologs were found using the protein BLAST tool present in the SEED database ( http://pubseed.theseed.org/ ).

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86 Figure A 2 Connection of arginine (Arg) biosynthesis to GTK and A m pathway. (A) Beginning with glutamate, ornithine is produced after five enzymatic steps. ArgA, N acetylglutamate synthase (EC 2.3.1.1); ArgB, Acetylglutamate kinase (EC 2.7.2.8); ArgC, N acetyl gamma glutamyl phosphate reductase (EC 1.2.1.38); ArgD, Acetylornithi ne aminotransferase (EC 2.6.1.11); ArgE, acetylornithine deacetylase (EC :3.5.1.16). (B) Examples of clustering of GTK and A m with Arg biosynthesis genes (in green) Genes in gray have functions unrelated to Arg Arrows point in the direction of transcri ption. Also listed is ArgG Argininosuccinate synthase (EC 6.3.4.5)

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87 Figure A 3. DAP lysine biosynthesis pathway (A) Variants of Lys biosynthesis (retrieved from Hudson et al., 2008). Listed enzymes are as follows: DapA, dihydrodipicolinate synthase (EC 4.2.1.52); DapB, dihydrodipicolinate reductase (EC 1.3.1.26); DapC, succinyldiaminopimelate aminotransferase (EC 2.6.1.17); DapD, L 2,3,4,5 tetrahydrodipicolinate succinyltransferase (EC 2.3.1.117); DapE, succinyldiaminopimelate desuccinylase (EC 3.5. 1.18); DapF, DAP epimerase (EC 5.1.1.7); LysA, m DAP decarboxylase (EC 4.1.1.20); Ddh, DAP dehydrogenase (EC 1.4.1.16); DapL, LL DAP aminotransferase (EC 2.6.1.83) (B) Examples of genomic clustering of Lys biosynthesis genes. Arrows point in the direction of transcription. Additional genes: LysC, Aspartokinase (EC 2.7.2.4) ; Asd, Aspartate semialdehyde dehydrogenase (EC 1.2.1.11 )

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88 Figure A 4. Phylogenetic analysis of GTK and variants of diaminopimelate aminotransferases. Sequences were aligned with ClustalW; the tree was constructed by the neighbor joining method (Saitou and Nei, 1987) with MEGA5. T he percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (F elsenstein, 1985) The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and P auling, 1965) and are in the units of the number of amino acid substitutions per site.

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89 A PPENDIX B CYCLIZATION OF KETOGLUTARAMATE The kinetic analysis of a midase ( Am ) enzymes presented in Chapter 2 had to take into account an important property of ketoglutaramate ( KGM ) In solution, KGM undergoes a reversible cyclization reaction to form a cyclic lactam (2 hydroxy 5 oxoproline) (Hersh, 1971; Meister, 1953). At neutral pH, the equilibrium heavily favors the cyclic form over the open chain form (99.7%:0.3%, respectively) (Hersh, 1971). Therefore, particularly at pH 8.0 or below, the rate of the Am enzymatic reaction is potentially limited by the rate of spontaneous hydrolysis of the predominant cyclic form because this form cannot be hydrolyzed by Am (Figure B 1) (Hersh, 1971; Jaisson et al., 2009; Krasnikov et al., 2009). All enzyme assays were performed at pH 8.5, where the rate of ring opening is less problematic (Hersh, 1971). However, even at this pH value the amount of Am used in assays must be carefully adjusted to ensure that the rate limiting step of the reaction is enzymatic and not the opening of the cyclic form. The appropriate amount of Am was determined by assaying KGM with a range of Am amounts (Figure B 2). The ideal result would be a doubling of the amount ketoglutarate ( KG) produced with the doubling of the reaction time. The quantity of Am used would be deemed too great if upon doubling the enzyme amount, the measured KG did not respond by a lso doubling. Given those criteria, the amount of tomato Am that is appropriate is 5 ng (Figure B 2A), and 10 ng for the maize enzymes (Figure B 2B). T Am ensured that the observed reaction rate was purely enzymatic and was not compl icated by the rate of ring opening.

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90 Fig ure B 1 The ketoglutaramate (KGM). The open chain form, KGM can be ketoglutarate, however, at neutral pH, KGM spontaneously cyclizes (99.7%) to form 2 hydroxy 5 oxoproline

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91 Figure B 2. Amidase ( Am) enzyme quantity validation assays. Varying amounts of Am ( GRMZM2G156486 ) were assayed. ketoglutarate as its 2,4 dinitrophenylhyd razone. Maize 1, GRMZM2G169365 had results similar to Maize 2 (data not shown). contained 50 mM Tris HCl, mercaptoethanol and 10 mM KGM.

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101 Taylor P P Pantaleone D P Senkpeil R F Fotheringham I G ., 1998 Novel biosynthetic approaches to the production of unnatural amino acids using transaminases. T rends Biotechnol 16 412 418 Van de Poel B, Bulens I, Oppermann Y, Hertog ML, Nicolai BM, Sauter M, Geeraerd AH. 2013. S adenosyl L methionine usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity. Physiol. Plant 148, 176 1 88. Vergara, F., Duffy, T.E., Plum, F., 1973. a Ketoglutaramate, a neurotoxic agent in hepatic c oma, Trans. Assoc. Am. Phys. 86, 255 263. Vinci C R Clarke S G ., 2010 Yeast, plants, worms, and flies use a methyltransferase to metabolize age damaged (R,S) AdoMet, but what do mammals do? Rejuvenation Res 13 362 364 Wamboldt, Y., Mohammed, S., Elowsky, C., Wittgren, C., de Paula, W.B.M., Mackenzie, S.A., 2009. Participation of leaky ribosome scanning in protein dual targeting by alternative translation initiation in higher plants. Plant Cell 21, 157 167. Wang Z Gerstein M Snyder M ., 2009 RNA Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10 57 63 Whetten R Sederoff R. 1995. Ligni n Biosynthesis. Plant Cell. 7, 1001 1013. Winge, I., McKinney, J.A., Ying, M., D'Santos, C.S., Kleppe, R., Knappskog, P.M., Haavik, J., 2008. Activation and stabilization of human tryptophan hydroxylase 2 by phosphorylation and 14 3 3 binding. Biochem. J. 410, 195 204. Yogev, O., Pines, O., 2011. Dual targeting of mitochondrial proteins: mechanism, regulation and function. Biochim. Biophys Acta 1808, 1012 1020. Zallot, R., Agrimi, G., Lerma Ortiz, C., Teresinski, H.J., Frelin, O., Ellens, K.W., Castegna, A., Russo, A., de Crcy Lagard, V., Mullen, R.T., Palmieri, F., Hanson, A.D., 2013. Identification of mitochondrial coenzyme a transporte rs from maize and Arabidopsis. Plant Physiol. 162, 581 588. Zhang, Q., Lee, J., Pandurangan, S., Clarke, M., Pajak, A., Marsolais, F., 2013. Characterization of Arabidopsis serine:glyoxylate aminotransferase, AGT1, as an asparagine aminotransferase. Phytoc hemistry 85, 30 35. Zhang, Q., Marsolais, F., 2014. Identification and characterization of omega amidase as an enzyme metabolically linked to asparagine transamination in Arabidopsis. Phytochemistry, Available online 22 January 2014.

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102 Zhong S Fei Z Chen Y R Zheng Y Huang M Vrebalov J McQuinn R Gapper N Liu B Xiang J Shao Y Giovannoni J J. 2013. Single base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening. Nat Biotechnol. 31 154 15 9. Zuckerkandl, E., Pauling, L., 1965. Evolutionary divergence and convergence in proteins. Edited in Evolving Genes and Proteins by V. Bryson and H.J. Vogel, pp. 97 166. Academic Press, New York.

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103 BIOGRAPHICAL SKETCH Kenneth William Ellens was born in Niagara Falls Ontario, Canada He attended Eden High School in St. Catharines, Ontario, where he graduated in 2003 In 2004 he attended Brock University as a Co op student in the biotechnology prog ram. This program allowed him to gain lab experience in multiple locations such as Agriculture and Agri Food Canada, in Vineland, Ontario, the Chemistry department at Brock University, as well as two labs in the National Research Council of Canada. The f irst was at the Plant Biotechnology Institute in Saskatoon, Saskatchewan and the second was at the Institute for Biological Sciences in Ottawa, Ontario. Ken completed his Honors thesis under the supervision of Dr. Vincenzo De Luca and Dr. Tony Yan. H e e arned his BSc. in biotechnology in the spring of 2009 In the spring of 2014 Ken earned his PhD. in Dr. Andrew Hanson at the University of Florida.



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4-Hydroxyphenylaceticacidderivativesofinositolfromdandelion ( Taraxacumof“cinale )rootcharacterisedusingLC…SPE…NMR andLC…MStechniques O.Kenny a b,T.J.Smyth a ,,C.M.Hewage b,N.P.Brunton c,P.McLoughlin a aDepartmentofFoodBiosciences,TeagascFoodResearchCentre,Ashtown,Dublin15,Ireland bConwayInstituteofBiomolecularandBiomedicalResearch,UCD,Bel“eld,Dublin4,Ireland cSchoolofAgricultureandFoodScience,UCD,Bel“eld,Dublin4,Ireland articleinfoArticlehistory: Received26August2013 Receivedinrevisedform4November2013 Availableonline17December2013 Keywords: Taraxacumof“cinale Dandelionroot 4-Hydroxyphenylaceticacidinositol LC…SPE…NMR LC…QTOF-MS/MS abstractThecombinationofhyphenatedtechniques,LC…SPE…NMRandLC…MS,toisolateandidentifyminorisomericcompoundsfromanethylacetatefractionof Taraxacumof“cinale rootwasemployedinthisstudy. Twodistinctfractionsof4-hydroxyphenylaceticacidderivativesofinositolwereisolatedandcharacterisedbyspectroscopicmethods.The 1HNMRspectraandMSdatarevealedtwogroupsofcompounds,one ofwhichwerederivativesofthedi-4-hydroxyphenylaceticacidderivativeoftheinositolcompoundtetrahydroxy-5-[2-(4-hydroxyphenyl)acetyl]oxycyclohexyl-2-(4-hydroxyphenyl)acetate,whiletheother groupconsistedofsimilartri-substitutedinositolderivatives.Forbothfractionsthederivativesofinositolsvaryinthenumberof4-hydroxyphenylaceticacidgroupspresentandtheirpositionandgeometry ontheinositolring.Intotal,threedi-substitutedandthreetri-substituted4-hydroxyphenylaceticacid inositolderivateswereidenti“edforthe“rsttimealongwithafurthertwopreviouslyreporteddi-substitutedinositolderivatives. 2013ElsevierLtd.Allrightsreserved. Introduction Dandelion( Taraxacumof“cinale )isaperennialherbofthe Asteraceae family.The Taraxacum genusconsistsofmanymicrospecies ( Hegi,1987 )andiswidespreadthroughoutmostofthenorthern hemisphere.Intraditionalandfolkmedicine,dandelionisrenownedforitsdiuretic,cholereticandlaxativeproperties( Bisset etal.,1994 ).Dandelionextractshavealsobeenshowntopossess anti-diabetic( Hussainetal.,2004;naletal.,2005 ),anti-carcinogenic( Babaetal.,1981;Kooetal.,2004 ),anti-in”ammatory( Tita etal.,1993;Yasukawaetal.,1996 ),antioxidant( Hagymasietal., 2000;HuandKitts,2005 )andprebiotic( Trojanovaetal.,2004 ) properties.Previouscharacterisationstudiesofextractsfromthe rootsof T.of“cinale haverevealedthepresenceofsesquiterpene lactones( Hanseletal.,1980;KisielandBarszcz,2000 ),anumber oftriterpenesandphytosterols( Akashietal.,1994;Burrowsand Simpson,1938;Furunoetal.,1993;Hanseletal.,1980 ),phenolic compounds( Williamsetal.,1996 ),particularlyhydroxycinnamic acids( Cliffordetal.,1987 )andcoumarins( Wolbisetal.,1993 ). ThehyphenationofHPLCwithNMRspectroscopyhasprovento beapowerfulanalyticaltoolthatcanbeusedinthestructureelucidationofcompoundsfromcomplexmixtures.Thishasgreatly diminishedtheneedtoisolateandpurifyindividualcompounds. ThefurthercouplingofLC…NMRtoonlinesolid-phaseextraction (SPE)hasconsiderablyimprovedthesensitivityofthistechnique byenablingtheisolatetobeconcentratedandelutedfullyindeuteratedsolvents.Asaresult,theidenti“cationofminorconstituentsfromcrudeextractsisnowpossibleinalesstimelymanner. Thepresentstudyfurtherinvestigateddandelionrootfornovel phenoliccompounds,whichresultedinthecharacterisationofa seriesofdiandtri-4-hydroxyphenylaceticacidinositolderivatives fromanethylacetateextractof T.of“cinale root,usingLC…SPE… NMRandLC…MS/MSspectroscopictechniques. Resultsanddiscussion NMRandLC…MS/MSanalysis Separationmethodsoffractions4and8weredevelopedusing HPLC.Atotalof5(compounds 1…5 )isolatesfromfraction4 ( Fig.1 )and3isolates(compounds 6…8 )fromfraction8( Fig.2 ) weretrappedseparatelyontheSPEcartridgesforfurtheranalysis byNMR.Thestructuresof5compoundsfromfraction4were determinedwiththeexceptionofpeak X ,whichappearedtobea0031-9422/$-seefrontmatter 2013ElsevierLtd.Allrightsreserved. http://dx.doi.org/10.1016/j.phytochem.2013.11.022 Correspondingauthor.Tel.:+353018059500. E-mailaddresses: owen.kenny@teagasc.ie (O.Kenny), thomas.smyth@teagasc.ie (T.J.Smyth), chandralal.hewage@ucd.ie (C.M.Hewage), nigel.brunton@ucd.ie (N.P.Brunton). Phytochemistry98(2014)197…203 Contentslistsavailableat ScienceDirectPhytochemistryjournalhomepage:www.elsevi er.com/locate/phytochem

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mixtureofco-eluteddi-4-hydroxyphenylaceticacidinositolsthat couldnotberesolvedundertheLCÂ…SPEÂ…NMRchromatographic conditionsused.The 1HNMRspectraofisolatesfromfractions 4and8showedbroadsimilaritiesthat,alongwithHPLCÂ… QTOF-MS/MSdata,suggestedarangeofderivativeswerepresent inbothofthesefractions.The 1HNMRspectrashowedclear evidenceofparadi-substitutedphenylgroupsconsistentwitha 4-hydroxyphenylaceticacidderivativeineachcase,whichisindicatedbythepresenceofdistinctsetsofdoublets( J =8.5Hz)inthe 7.2and6.8ppmregionsofthespectra.Incompounds 1Â…5 theintegrationsuggestedtwo4-hydroxyphenylaceticadducts,whilethe integrationofcompounds 6Â…8 suggestedthepresenceofthree 4-hydroxyphenylaceticacidadducts.Theremainderofthesignals suggestedaninositoltypestructure,wheretheprotonsoncarbons Fig.1. LCÂ…SPEÂ…NMRchromatogramoffraction4showingthetrappedcompounds 1Â…5 .Peak X representsamixtureofco-elutedcompounds,whichwereunresolved spectroscopically. Fig.2. LCÂ…SPEÂ…NMRchromatogramoffraction8showingthetrappedcompounds 6Â…8 198 O.Kennyetal./Phytochemistry98(2014)197Â…203

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with4-hydroxyphenylaceticacidadductsattachedareshifted down“eld,comparedtothoseatunsubstitutedpositions,giving signalsbetween5.5and4.5ppm.Ingeneral,thesplittingpatterns andconstantsforthesepeaksallowedforthedistinctionbetween axialandequatorialsubstitutionpatternsontheinositolring.This alsoappliesfortheadditionalnon-substitutedportions,allowing fortheorientationateachpointoftheinositolringtobedetermined.Acleardistinctioncanbemadeusingthesplittingpatterns withinthe5.0…5.5region,whereasplittingof10Hzsuggestedan equatorialpositionofthesubstituent(andthereforeaxialposition ofthehydrogen).Meanwhile,splittingbelow4Hzsuggestedan axialpositionofthesubstituent(andthereforeequatorialposition ofthehydrogen)oranequatorialpositionofthesubstituentwith onlyequatorialhydrogensadjacent.Incaseswhereaparticularaxialhydrogenwasadjacenttobothanaxialandanequatorial hydrogentheresultingpeakwasadoubledoublet,withsplitting of10and4Hz( Gaoetal.,1990 ).Intheabsenceofcomplications duetosymmetryofthemoleculesthesplittingpatternsofadjacent protonsallowthedistinctionstobemadebetweenaxial…equatorialandequatorial…equatorialsplittinginallofthecompounds shown.Thesplittingpatternsofallofthesesignals,combinedwith thecrosspeaksfromtheCOSYandTOCSYspectraallowedforthe determinationof4-hydroxyphenylaceticadductconnectivityand geometryaroundthering.HPLC…QTOF-MS/MSaccuratemassmeasurementwasusedinconjunctionwith 1HNMRtocon“rmthe structuralcharacterisationofeachcompound. Thesymmetryofcompounds 1 and 2 gavetheappearanceof lesscomplexspectrathanwasexpected.Thishasbeenpreviously notedinthecharacterisationofinositolderivativesfromasub-aerialmethanolextractof Taraxacumlinearisquameum ( Zidornetal., 1999 ),wherethesymmetricalnatureofthemoleculegaveriseto aspectrumwithdoubletsfor4-hydroxyphenylaceticmoietyand threesingletsfortheinositoltypemoiety.Compound 3 wasalso similartoonedescribedby Zidornetal.(1999) andwasassigned accordingly.Thesubstitutedpositionsshowedatripletat 5.13ppmandadoubledoubletwith J =10and3Hzat4.83ppm, showingthatthehydrogenatthe4.83ppmwasaxialwithanaxial andanequatorialhydrogenadjacent.Thepeaksat3.52ppmand 3.76ppmaretriplets(withsomeadditionalOHsplitting)andare thereforeaxialsurroundedbyaxialhydrogens.Thepeakat 3.85ppmisadoubledoubletsuggestinganaxialhydrogenwith anaxialandanequatorialhydrogenadjacentwhilethepeakat 3.99ppmisduetoanequatorialhydrogen.Thisgivesrisetothe structureshown.Compound 4 showedclearlinkagesinCOSYspectra,whichindicatedthatthesubstitutedpositionswereadjacentto eachother.ThesplittingpatternsandCOSYspectrawereusedto determinethegeometrywiththeprotonat5.23ppmbeingatripletwithsplitting J =10Hzsuggestinganaxial…axial…axialpattern andat5.03ppmadoubledoubletsuggestinganaxial…axial…equatorialgeometry.Thepeaksat3.69ppmand3.73ppmweredeterminedtobeaxialwhile3.95ppmand4.06ppmbeingequatorial protons.Compound 5 showedamorecomplicatedpatternthan compounds 1 and 2 thoughwithsomepeakscoinciding.The substitutedpeakat5.17ppmshowssplittingof J =3.8Hz,while thatat5.02ppmshowsasplittingof J =10Hz.Thissuggestedan axialpositionforthepeakat5.02ppm,withtwoadjacentaxial protonswhile5.17ppmiseitheraxialsurroundedbyequatorial protonsorequatorialwitheitherequatorialoraxialprotons.The remaininginositolprotonsshowtwogroupsof2protonswithaxialandequatorialsplittingpatterns.Thepeakat3.61ppmisatripletsuggestingthatthesurroundingprotonsareintheaxial position.Thepeakat3.88ppmgivesaslightlydistorteddouble doublet,suggestingthattheprotonsareaxialandareadjacentto oneequatorialandoneaxialproton,givingrisetothestructure shown.TheTOCSYspectraandcrosslinkedpeaksintheCOSY spectracon“rmedthesepatterns. Theaccuratemassmeasurementofdi-4-hydroxyphenylacetic acidinositols,compounds 1…5 ,showedanelementalcomposition ofC22 H23 O10 ([M H] observed m / z 447.1289,calculated m / z 447.1291).Fragmentationofthesederivatives( Fig.3 )resultedin twomajorproductions m / z 295.1023(C14 H15 O7 )and m / z 151.0509(C8 H7 O3 ).Thesefragmentscorrespondedtothemono 4-hydroxyphenylaceticacidinositoland4-hydroxyphenylacetic acidadductsrespectively.ThecombinationofNMRandLC…MS/ Fig.3. HPLC…MS/MSspectrumshowingthefragmentationpatternofadi-4-hydroxyphenylaceticacidinositol(compound 1 ).Theannotationarepresentsanaxial orientationofthesubstituent. O.Kennyetal./Phytochemistry98(2014)197…203 199

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MSdatacon“rmedthestructuresofdi-4-hydroxyphenylacetic acidinositolderivativesasfollows;compounds 1 2 and 5 as1,4 substituted,compound 3 as1,3substitutedandcompound 4 as 1,2substituted( Fig.4 ).Inositolwasusedasadescriptorforthe rangeofstructuresfound,asamixtureofvariousisomerswas present.Compounds 3 4 ,arechiro-inositols,compound 1 isascyllo-inositol,compound 2 isaneo-inositolandcompound 5 isa muco-inositol. Forisolatesoffraction8,peaksrelatingtothe4-hydroxyphenylaceticportionsofeachmoleculefrequentlyoverlapped,giving risetobroaddoubletsandtheappearanceofdoubledoublets.In eachcaseanintegrationof6forpeaksdown“eldof7ppmand6 forpeaksup“eldof7ppmwasconsistentwithourproposed structures.Theinositolportionofthestructurewasdivisibleinto portionsadjacenttoanOHgroupandportionswitha4-hydroxyphenylaceticsubstituent,withdown“eldshiftsasdescribedabove forcompounds 1…5 .Forcompounds 6…8 ,aclearpatternofprotons withvariedsplittingforthreeprotonswithinthe4.5…5.5ppmregionswasseen,thusallowingthegeometryofthesubstituents, includingthehydroxylsubstituents,inthesecompoundstobe determinedandwascon“rmedbyCOSYspectra.Forcompound 6 ,COSYspectrashowthatthesubstituentsareonadjacentcarbons.Ofthesetheprotongivingapeakat5.21ppmisatripletof J =10Hzandthereforemustbeinanaxialpositioninteracting withtwoaxialprotons.Theprotongivingapeakat5.12ppmas adoubledoubletwith J =10and3.4mustbeaxialinteractingwith anaxialandequatorialproton,leavingtheprotongivingrisetoa peakat5.28ppmasequatorial.Thepeakat3.74ppmshowssplittingpatternscharacteristicofbeinganaxialprotoninteracting withtwoaxialprotons,whilethatat3.65ppmbeinganaxialprotoninteractingwithanaxialandequatorialproton.Thecombinationofthisandsplittingpatternsshowthepeakat3.93ppmtobe duetoanequatorialproton. Compound 7 showsoneprotonat4.9ppmwhichisaxialwith oneaxialandoneequatorialadjacentproton.Theremaining substitutedpositionsshownosignofaxial-axialsplittingandare adjacentfromtheCOSY,thesplittingpatternsandcoincidingof thephenylgroupssuggestthattheyareinthesamecon“guration andareequatorial.Theprotonatposition5and3areassignedas axialandequatorialrespectivelyduetothesplittingatposition2 withthe“nalprotonatposition2beingassignedasaxial. Inthecaseofcompound 8 ,theCOSYrevealsthattwoofthesubstitiuentsareadjacentwiththeremainingpeakisolatedfromthese by1position.Thesubstitutedpeaksconsistoftwogivingdouble doublets,with J =10and3Hz,withtheremainingpeakbeinga tripletof J =3.4Hz.Thisimpliesanaxialhydrogenfortwoofthe substituentswithanadjacentaxialandequatorialhydrogenfor eachcase.TheOHsubstitutedpositionsgivetripletssuggesting interactionsbetweenaxialprotonsonlyfortwopositions,while theremaining4.01ppmpositionmustbeinanequatorialcon“guration.Theprotongivingrisetothepeakat5.26ppmmustalsobe inanequatorialpositionasanaxialhydrogenwouldalsogivea doubledoubletinthisposition.Forcompounds 6…8 thepeakscorrespondingtopositions2and6ofthephenylmoietyalsogavedistinctdifferencesbetweenaxialandequatorialpositions,which correspondedtothesubstitutionpatterndetermined. Theaccuratemassesoftri-4-hydroxyphenylaceticacidinositols, compounds 6…8 ,showedanelementalcompositionofC30 H29 O12([M H] observed581.1646,calculated581.1659).Asimilar fragmentationpattern( Fig.5 )tothedi-4-hydroxyphenylaceticacid Fig.4. Chemicalstructuresofdi-4-hydroxyphenylaceticinositols(compounds 1…5 )isolatedfrom Taraxacumof“cinale usingLC…SPE…NMR.Theannotationarepresentsan axialorientationanderepresentsanequatorialorientationofthesubstituent. 200 O.Kennyetal./Phytochemistry98(2014)197…203

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inositolswasobservedforthesecompounds,wherethesuccessive lossof4-hydroxyphenylaceticacidadductsproducedamono4hydroxyphenylaceticacidinositol.AnalysisofbothNMRandLC… MS/MSdatacon“rmedcompound 6 asbeing1,2,3substituted,compound 7 as1,2,4substitutedandcompound 8 as1,2,5substituted tri-4-hydroxyphenylaceticacidchiro-inositolderivatives( Fig.6 ). Conclusion Theisolationandstructuralelucidationofdi-andtri-4hydroxyphenylaceticacidderivativesofinositolfrom T.of“cinale demonstratestheusefulnessofLC…SPE…NMRfornaturalproduct identi“cation.LC…SPE…NMRwasusedtoisolate“vecompounds fromfraction4andthreecompoundsfromfraction8respectively. Furtherinvestigationby 1HNMRandHPLC…QTOF-MS/MSrevealed thepresenceof5di-4-hydroxyphenylaceticacidinositolderivatives(compounds 1…5 )fromfraction4ofwhichcompounds 1 4 and 5 arereportedforthe“rsttime( Fig.4 ).Inaddition,3previouslyunreportedtri-4-hydroxyphenylaceticacidinositolderivatives(compounds 6…8 )werecharacterisedfromfraction8( Fig.6 ). Experimental Generalexperimentalprocedures HPLCgrade n -hexane,DCM,EtOAc,MeOH,MeCN,andH2 Owere purchasedfromFisherScienti“cLtd.(Loughborough,Leicestershire,UK).MgSO4 andLC…MSgradeFAwereobtainedfromSigma…Aldrich(Wicklow,Ireland).LC…SPE…NMRwasperformedon asystemconsistingofanAgilent1200HPLC(AgilentTechnologies, GmBH,Gerrmany)“ttedwithaBrukerDADdetector(BrukerUK Ltd.,Coventry,UK),SparkProspekt2system(SparkHollandBV, Emmen,Holland)andGilsonliquidhandler(GilsonInc.Middleton, WI,USA).HySphere’Polydivinyl-benzeneresin“lledSPE cartridges(10 2mm,10…12lm)(SparkHollandBV,Emmen, Holland)wereusedtotrapresolvedpeaksfromfractions.NMR analysiswascarriedoutusinga1.7mmTXIprobeandaBruker AvanceIII500MHzspectrometer(BrukerUKLtd.,Coventry,UK) usingCD3 CNasasolventat25 C.LC…MSanalysiswasperformed onaWatersAlliance2695HPLCsystemcoupledtoaWatersQ-Tof PremiermassspectrometerusinganAgilentC18 column (2.1 100mm,2.7lm).UPLC…MS/MSwascarriedoutonaWaters AcquityUPLCsystemcoupledtoaWatersAcquitytandemmass spectrometer(TQD)usingaWatersAcquityUPLCHSST3column Fig.5. HPLC…MS/MSspectrumshowingthefragmentationpatternofatri-4-hydroxyphenylaceticacidinositol(compound 6 ).Theannotationarepresentsanaxial orientationofthesubstituent. Fig.6. Chemicalstructuresoftri-4-hydroxyphenylaceticinositols(compounds 6…8)isolatedfrom Taraxacumof“cinale usingLC…SPE…NMR.Theannotationa representsanaxialorientationofthesubstituent. O.Kennyetal./Phytochemistry98(2014)197…203 201

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(2.1 100mm,1.7lm)withaWatersC18 VanGuard(5 2.1mm, 1.8lm). Plantmaterial DandelionrootswerepurchasedfromIrishOrganicHerbsLtd. (Drumshanbo,Co.Leitrim,Ireland).Avoucherspecimen (DBN27:2013)ofdandelionwasdepositedintheHerbariumof theNationalBotanicGardens(Dublin,Ireland).Thesamplewas subsequentlyidenti“edbyatrainedbotanistasthemicrospecies T.of“cinale F.H.Wigg.Therootswerethenthinlysliced (<10mm)andfreezedried(A12/60FreezeDryer,FrozenInTime Ltd.,York,England).Thedriedrootswereblended(Waring CommericalBlender,ChristisonParticleTechnologies,Gateshead, UK)intoa“nepowderandstoredat…80 Cuntilfurtheruse. Isolation Freezedrieddandelionrootpowder(4kg)wasextractedusing excessvolumesofDCMbeforebeingexhaustivelyextractedwith MeOHusingaMaxQ6000shaker(ThermoScienti“c,Iowa,USA) atroomtemperature.TheMeOHextractwasdriedat40 Cusing arotaryevaporator(Laborata4000Ef“cient,HeidolphLtd.,Germany).SolventpartitioningofthedriedMeOHextract(600g) wascarriedoutby“rstdissolvingtheextractinH2 Oandthen washingrepeatedlywithethylacetate(EtOAc)inaseparatingfunnel.TheEtOAcextractwaspassedthroughmagnesiumsulphate (MgSO4 ),undervacuumusingaBuchnerfunnel,toremovetraces ofH2 O.Theextractwasthendriedat40 Cusingarotaryevaporator.FractionationoftheEtOAcfraction(16g)wascarriedoutona VarianIntelliFlash310FlashChromatography(AnalogixSemiconductorInc.,California,US)systemusinganAgilentSuperFlash’ SF40…240gnormalphasecolumn(40.6mm 37.9cm,50lm). Fractionswereelutedwith n -hexane…EtOAcmixtures,startingat 0%EtOAc(upto100%EtOAc)instepwise10%incrementsofEtOAc. Atotalof11fractionsweredriedat40 Cusingarotaryevaporator. Fraction10(1.67g)( n -hexane…EtOAc,10:90)wasfurtherfractionatedusinganAgilentSuperFlash’SF40…300gC18 column (40.6mm 34.8cm,50lm)withH2 Oand3%incrementsof MeOH.Atotalof12fractionswerecollected.MeOHwasremoved usingarotaryevaporatorat40 Cpriortofreezedrying.Fraction4 (25.8mg),elutedwithH2 O…MeOH(91:9),and8(24.4mg),with H2 O…MeOH(79:21),wereselectedforfurtheranalysis. LC…SME…NMR SeparationandisolationforNMRoffractions4and8was achievedusinganAgilentEclipseXDB-C18 column (4.6mm 150mm,5lm).Forfraction4,abinarysolventsystem ofH2 O(mobilephaseA)andMeCN(mobilephaseB)wasusedas follows;0…5min:2…7%B,5…23min:7…12%B,23…27min: 12…98%B.Inthecaseoffraction8,abinarysolventsystemof H2 O(mobilephaseA)andMeCN(mobilephaseB)wasalsoutilised asfollows;0…2min:2%B,2…6min:2…15%B,6…21min:15…25%B, 21…24min:25…98%B,24…27min:98%B,27…29min:98…2%B, 29…30min:2%B.Thecolumnoventemperaturewassetat25 C andthewavelengthoftheDADdetectorwassetto226nmforboth fractions.HySphere’Polydivinyl-benzeneresin“lledSPEcartridgeswereusedtotrapresolvedpeaksfromfractions.Thedrying timeforeachcartridgewas1husingnitrogengas.Metabolites wereelutedfromcartridgesusing50llofCD3 CNintoseparate 1.7mmborosilicateNMRtubesusingaGilsonliquidhandler platform. NMRanalysiswascarriedoutonalltrappedisolatesfromfractions4and8.ThespectrawerereferencedtotheresidualCD3 CN signal.Duetothepresenceofresidualsolvent,a1DNOESYusing doublepre-saturationduringrelaxationdelayandmixingtime wasusedtoacquirethespectra.Atotalof32kdatapointswererecordedoverasweepwidthof10,000Hz,with512scans.Anexponentiallinebroadeningof1Hzwasimposedontheaccumulated databeforeFouriertransformation.Structureswerecon“rmed usingCOSYandTOCSYsequences. HPLC…MS/MSanalysis Sampleswerediluted1in2withMeOHandtransferredfrom NMRtubestostandard2mlsamplevialscontainingaglassinsert. MobilephaseA(H2 O+0.1%FA):mobilephaseB(MeCN+0.1%FA) wereusedforchromatographicseparationasfollows;0…2min:2% B,2…14min:2…98%B,14…19min:98%B,19…21min:98…2%B, 21…25min:2%B.Thecolumnoventemperaturewassetat50 C. Q-TOFMSwasconductedinnegativemodeESI.Thesourcetemperaturewas120 Candthedesolvationtemperaturewas300 C. Ineachcase,nitrogenwasusedasthedesolvationgas(800L/h) andconegas(50L/h).Theconevoltagewas40volts,whilethelock masswassettoLeucineEnkephalin[M+H] +(556.2771 m / z ).Sampleswereanalysedinnegativemodeintherange m / z 150…1200. Compound1: UV(MeCN:H2 O): kmax 191,222and275nm. 1H NMRspectraldata(500MHz,CD3 CN) d H7.22,(4H, d J =7.9Hz, C2 0,2 00,6 06 00-H),6.86(4H, d J =7.9Hz,C3 0,3 00,5 05 00-H),5.00(2H, m C1-H,C4-H),4.06(2H, bs ,C3-H,C5-H),4.02(2H, bs ,C2-H,C6-H), 3.71(2H, s ,C b 0-H),3.70(2H, s ,C b 00-H).ESI-Q-TOF-MSobserved m / z 447.1289[M H] (calculated m / z 447.1291,C22 H23 O10 ). Compound2: UV(MeCN:H2 O): kmax 191,222and275nm. 1H NMRspectraldata(500MHz,CD3 CN) d H7.20(4H, d J =8.5Hz, C2 0,2 00,6 06 00-H),6.86(4H, d J =8.5Hz,C3 0,3 00,5 0,5 00-H),5.01(2H, s C1-H),3.66(4H, s ,C b 0-H,C b 00-H),3.39(2H, bs ,(C2-H,C6-H),3.33 (2H, s ,C3-H,C5-H).ESI-Q-TOF-MSobserved m / z 447.1289[M H] (calculated m / z 447.1291,C22 H23 O10 ). Compound3: UV(MeCN:H2 O): kmax 205,222and275nm. 1H NMRspectraldata(500MHz,CD3 CN) d H7.23…7.18(4H, dd J =8.5Hz,C2 0,2 00,6 06 00-H),6.87…6.81(4H, dd J =8.5,C3 0,3 00,5 0,5 00-H), 5.13(1H, t J =3.6Hz,C1-H),4.84(1H, dd J =10,3Hz,C3-H),4.00 (1H, dd J =3,4Hz,C2-H),3.85(1H, dd J =3,4Hz,C6-H),3.76 (1H, dt J =10,4Hz,C4-H),3.69(2H, s ,C b 0-H),3.67(1H, s ,C b 00-H), 3.51(1H, dt J =10,4Hz,C5-H).ESI-Q-TOF-MSobserved m / z 447.1289[M H] (calculated m / z 447.1291,C22 H23 O10 ). Compound4: UV(MeCN:H2 O): kmax 191,222and275nm. 1H NMRspectraldata(500MHz,CD3 CN) d H7.16…7.11(4H, dd J =8.5Hz,C2 0,2 00,6 06 00-H);6.85…6.83(4H, dd J =8.5Hz,C1-H, C3 0,3 00,5 0,5 00-H),5.24(1H, t J =10Hz,CH-1),5.07(1H, dd J =10, 3Hz,CH2),4.06(1H, q J =3.5Hz,C3-H),3.95(1H, mJ =3.5Hz, C4-H),3.70(2H, m J =10,3.5Hz,C5-H,C6-H),3.53(2H, s ,C b 0-H), 3.47(2H, s ,C b 00-H).ESI-Q-TOF-MSobserved m / z 447.1289[M H] (calculated m / z 447.1291,C22 H23 O10 ). Compound5: UV(MeCN:H2 O): kmax 205,224and277nm. 1H NMRspectraldata(500MHz,CD3 CN) d H7.24…7.19(4H, dd J =8.5, C2 0,2 00,6 06 00-H),6.86(4H, d J =8.5,C1-H,C3 0,3 00,5 0,5 00-H),5.17(1H, t J =3.8Hz,C1-H),5.02(1H, t J =10Hz,C4-H),3.89(2H, m J =3.8Hz,C2-H,C6-H),3.70(2H, s ,C b 0-H),3.67(2H, s ,C b 00-H), 3.60(2H, dd J =10,3.6Hz,C3-H).ESI-Q-TOF-MSobserved m / z 447.1289[M H] (calculated m / z 447.1291,C22 H23 O10 ). Compound6: UV(MeCN:H2 O): kmax 197,222and275nm. 1H NMRspectraldata(500MHz,CD3 CN) d H7.99(4H, dd J =8.5Hz, C2 00,2 000,6 00.6 000-H),6.98,(2H, d J =8.5Hz,C2 0,6 0-H),6.85(4H, d J =8.5Hz,C3 00,3 000,5 00,5 000-H),6.90(2H, d J =8.5Hz,C3 0,5 0-H),5.28 (1H, t J =3.8Hz(C1-H),5.22(1H, t J =10Hz,C3-H),5.12(1H, dd J =10,3.8Hz,C2-H),3.93(1H, q J =3.8Hz(C6-H),3.76(1H, dt J =10,4Hz,C4-H),3.65(1H, dd J =10,3Hz,C5-H),3.61(2H, s C b 0-H),3.60(2H, s ,C b 00-H),3.52(2H, s ,C b 000-H).ESI-Q-TOF-MSobserved m / z 581.1646[M H] (calculated m / z 581.1646, C30 H29 O12 ). 202 O.Kennyetal./Phytochemistry98(2014)197…203

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Compound7: UV(MeCN:H2 O): kmax 191,224and277nm. 1H NMRspectraldata(500MHz,CD3 CN) d H7.20(4H, dd J =8.5Hz, C2 0,2 00,6 0,6 00-H),7.09(2H, d J =8.5Hz,C2 000,6 000-H),6.86(2H, d J =8.5Hz,C3 0,3 00,5 0,5 00-H),6.82(4H, d J =8.5Hz,C3 0,3 00,3 000,5 0,5 00,5 000-H),5.23(1H, t J =3.8Hz(C1-H),5.14(1H, t J =3.8Hz(C2-H),4.88(1H, dd J =10,3Hz(C4-H),3.73(1H, m J =10,3Hz(C3-H),3.67(2H, s ,C b 0-H),3.65(1H, m ,C5-H),3.63 (2H, s ,(C b 00-H),3.61,2H, s ,(C b 000-H)3.65,1H, m ,C5-H),3.54(1H, m, C6-H).ESI-Q-TOF-MSobserved m / z 581.1646[M H] (calculated m / z 581.1646,C30 H29 O12 ). Compound8: UV(MeCN:H2 O): kmax 190,222and275nm. 1H NMRspectraldata(500MHz,CD3 CN) d H7.22,(4H, bt J =8.5Hz, C2 00,2 000,6 00,6 000-H)7.11(2H, d J =8.5Hz,C-2 0,6 0-H),6.87…6.81(6H m ,C3 0,3 00,3 000,5 0,5 00,5 000-H),5.25(1H, t J =3.4Hz,C1-H);5.04(1H, dd J =10,3.4Hz,C2-H),4.90(1H, dd J =10,3.4Hz,C5-H),4.03 (1H, q J =3.4Hz,C6-H),3.84(1H, dt J =10,4Hz,C4-H);3.73… 3.68, m J =10,3.4Hz,C3-H),3.71,(2H, s ,C b 0-H),3.63(2H, s ,C b 00H),3.62(2H, s ,C b 000-H).ESI-Q-TOF-MSobserved m / z 581.1646 [M H] (calculated m / z 581.1646,C30 H29 O12 ). Acknowledgements TheauthorswouldliketoacknowledgetheIrishPhytochemical andFoodNetwork(IPFN)andTeagascWalshFellowshipscheme fortheir“nancialandtechnicalsupport.Theauthorswouldalso liketothankDr.ColinT.KelleherfromtheNationalBotanicGardens,Dublinforhisidenti“cationofthedandelionspecies. AppendixA.Supplementarydata Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,at http://dx.doi.org/10.1016/j.phytochem.20 13.11.022 References Akashi,T.,Furuno,T.,Takahashi,T.,Ayabe,S.I.,1994.Biosynthesisoftriterpenoidsin culturedcells,andregeneratedandwildplantorgansof Taraxacumof“cinale Phytochemistry36,303…308 Baba,K.,Abe,S.,Mizuno,D.,1981.Antitumoractivityofhotwaterextractof dandelion, Taraxacumof“cinale …correlationbetweenantitumoractivityand timingofadministration(authorstransl.).J.Pharm.Soc.Jpn.101,538…543 Bisset,N.G.,Phillipson,J.D.,Czygan,F.C.,Frohne,D.,Holtzel,D.,Nagell,A.,1994. HerbalDrugsPhytopharmaceuticals:AHandbookforPracticeonaScienti“c Basis.CRCPress,BocaRaton,AnnArbor,London,Tokyo Burrows,S.,Simpson,J.,1938.Thetriterpenealcoholsof Taraxacum root.The triterpenegrouppartIV.J.Chem.Soc.2,2042…2047 Clifford,M.N.,Shutler,S.,Thomas,G.A.,Ohiokpehai,O.,1987.Thechlorogenicacids contentofcoffeesubstitutes.FoodChem.24,99…107 Furuno,T.,Kamiyama,A.,Akashi,T.,Usui,M.,1993.Triterpenoidconstituentsof tissueculturesandregeneratedorgansof Taraxacumof“cinale .PlantTissueCult. Lett.10,275…280 Gao,F.,Wang,H.,Marby,T.J.,1990.Inositolderivativesandpseudoguainolidesfrom Hymenoxystexana .Phytochemistry29,2273…2276 Hagymasi,K.,Blazovics,A.,Feher,J.,Lugasi,A.,Kristo,S.T.,Kery,A.,2000.The invitro effectofdandelionsantioxidantsonmicrosomallipidperoxidation.Phytother. Res.14,43…44 Hansel,R.,Kartarahardja,M.,Huang,J.T.,Bohlmann,F.,1980.Sesquiterpenlactonb -D-glucopyranosidesowieeinneueseudesmanolidaus Taraxacumof“cinale Phytochemistry19,857…861 Hegi,G.,1987.IllustrierteFloravonMitteleuropa.CompositaeII,.2nded.2nded., vol.4PaulParey,Berlin,Hamburg Hussain,Z.,Waheed,A.,Qureshi,R.A.,Burdi,D.K.,Verspohl,E.J.,Kahn,N.,Hasan,M., 2004.TheeffectofmedicinalplantsofIslamabadandMureeregionofPakistan oninsulinsecretionfromINS-1cells.Phytother.Res.18,73…77 Hu,C.,Kitts,D.D.,2005.Dandelion( Taraxacumof“cinale )”owerextractsuppress bothreactiveoxygenspeciesandnitricoxideandpreventslipidoxidation invitro .Phytomedicine12,588…597 Kisiel,W.,Barszcz,B.,2000.Furthersesquiterpenoidsandphenolicsfrom Taraxacum of“cinale .Fitoterapia76,269…273 Koo,H.N.,Hong,S.H.,Song,B.K.,Kim,C.H.,Yoo,Y.H.,Kim,H.M.,2004. Taraxacum of“cinale inducescytotoxicitythroughTNF-aandIL-1asecretioninHepG2 cells.LifeSci.74,1149…1157 nal,S.,Timur,S.,Okutucu,B.,Zihniog lu,F.,2005.Inhibitionofa-glucosidaseby aqueousextractofsomepotentantidiabteicmedicinalherbs.Prep.Biochem. Biotechnol.35,29…36 Tita,B.,Bello,U.,Faccendini,P.,Bartolini,R.,Bolle,P.,1993. Taraxacumof“cinale W.: pharmacologicaleffectofethanolextract.Pharmacol.Res.27,23…24 Trojanova,I.,Rada,V.,Kokoska,L.,Vlkova,E.,2004.Thebi“dogeniceffectof Taraxacumof“cinale root.Fitoterapia75,760…763 Williams,C.A.,Goldstone,F.,Greenham,J.,1996.Flavonoids,cinnamicacidsand coumarinsfromthedifferenttissuesandmedicinalpreparationsof Taraxacum of“cinale .Phytochemistry42,121…127,http://www.ncbi.nlm.nih.gov/pubmed/ 8728061 Wolbis,M.,Krolikowska,M.,Bednarek,P.,1993.Phenoliccompoundsin Taraxacum of“cinale .ActaPol.Pharm.50,153…158 Yasukawa,K.,Akihisa,T.,Oinuma,H.,Kasahara,Y.,Kimura,Y.,Yamanouchi,S., Kumaki,K.,Tamura,T.,Takido,M.,1996.Inhibitoryeffectofdi-andtrihydroxy triterpenesfromthe”owersof Compositae on12O -tetradecanoylphorbol-13acetateinducedin”ammationinmice.Biol.Pharm.Bull.19,1329… 1331 Zidorn,C.,Ellmerer-Muller,E.P.,Stuppner,H.,1999.Eudesmanolidesandinositol derivativesfrom Taraxacumlinearisquameum .Phytochemistry51,991… 994 O.Kennyetal./Phytochemistry98(2014)197…203 203