Isolation and characterization of KDOP synthase and two isozymes of DAHP synthase in Spinacia oleracea L. and Solanum tu...

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Title:
Isolation and characterization of KDOP synthase and two isozymes of DAHP synthase in Spinacia oleracea L. and Solanum tuberosum L.
Physical Description:
ix, 93 leaves : ill. ; 28 cm.
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English
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Doong, Ron Lou, 1949-
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Subjects / Keywords:
Plant enzymes   ( lcsh )
Chromatographic analysis   ( lcsh )
Spinach   ( lcsh )
Potatoes   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1990.
Bibliography:
Includes bibliographical references (leaves 86-92).
Statement of Responsibility:
by Ron Lou Doong.
General Note:
Vita.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
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notis - AJD0502
oclc - 25682009
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Full Text





ISOLATION AND CHARACTERIZATION OF KDOP SYNTHASE AND TWO
ISOZYMES OF DAHP SYNTHASE IN Spinacia oleracea L. AND
Solanum tuberosum L.


















By

RON LOU DOONG


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


1990































Copyright 1990

by

Ron Lou Doong













ACKNOWLEDGEMENTS

I offer my sincere gratitude to Dr. Roy Jensen who has

provided financial support, help, and guidance throughout my

endeavor to accomplish my goal in science. I am very much

indebted to Dr. John Gander for his invaluable instructions on

carbohydrate chemistry. Special thanks are also due to Dr. K.

T. Shanmugam, Dr. P. Chun, Dr. D. Duggan, and Dr. W. Gurley

for their advice while serving on my advisory committee.

I also wish to thank Dr. Randy Fischer, Dr. Tianhui Xia,

Ms. Carol Bonner, and Mrs. Premila Rao for their friendly help

and my fellow graduate students Prem Subramaniam and Genshi

Zhao for the sharing of their wisdom. I owe my son Larry, my

friend Ms. Eunice Johnson, Mr. Chris Fredette, Mr. Yaw-Hwa

Liou and Dr. Simon Wang very much for their patient help with

the proofreading and preparation of this dissertation.


iii














TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS.............................. iii

LIST OF FIGURES. ........................... .... v

LIST OF TABLES................................... vi

ABSTRACT ................................ vii

CHAPTER

I LITERATURE REVIEW AND RATIONALE...... 1

II DAHP SYNTHASE-Mn (DS-Mn)............. 6

INTRODUCTION........................ 6
MATERIALS AND METHODS.................. 7
RESULTS............... ..... ......... 10

III DAHP SYNTHASE-Co (DS-Co)............. 44

INTRODUCTION........................ 44
MATERIALS AND METHODS............... 44
RESULTS. ............ .......... ......... 46
DISCUSSION ............... ........ 59

IV KDOP SYNTHASE........................ 66

INTRODUCTION......................... 66
MATERIALS AND METHODS................ 67
RESULTS..................... ......... 69
DISCUSSION .......................... 85

REFERENCES...... .......................... ..... 86

BIOGRAPHICAL SKETCH........................ ....... 93













LIST OF FIGURES


2-1 Elution profiles of potato DS-Mn, DS-Co, and KDOP
synthase from DEAE cellulose...................... 13

2-2 Elution profiles of spinach DS-Mn, DS-Co, and KDOP
synthase from DEAE cellulose....................... 15

2-3 pH optima of DS-Mn and DS-Co from spinach.......... 19

2-4 Temperature optima of DS-Mn and DS-Co from spinach. 21

2-5 Divalent metal effects on spinach DS-Mn............ 24

2-6 Effects of DTT and thioredoxin on spinach DS-Mn.... 26

2-7 Progress curve of fully activated spinach DS-Mn.... 28

2-8 Saturation curve of spinach DS-Mn by PEP........... 34

2-9 Saturation curve of spinach DS-Mn by E4P........... 36

2-10 Inhibiton curve of spinach DS-Mn by arogenate...... 38

2-11 Induction of DS-Mn and DS-Co in potato tubers by
mechanical wounding ................................ 40

3-1 Divalent metal effects on spinach DS-Co............ 51

3-2 Saturation curves of spinach DS-Co by PEP with
various cosubstrates .............................. 53

3-3 Kinetics of periodate oxidation of enzymatic
products of spinach DS-Co with various substrates.. 57

4-1 Temperature optimum of spinach KDOP synthase....... 77

4-2 pH optimum of KDOP synthase from spinach........... 79

4-3 Thin-layer chromotography of KDOP and KDO.......... 80

4-4 Kinetics of oxidation of KDOP by periodate......... 82

4-5 Double reciprocal plot for arabinose-5-phosphate... 84













LIST OF TABLES
PaUe

2-1 Thermostability of spinach DS-Mn................... 16

2-2 Effect of DTT and/or thioredoxin on DS-Mn activity
from crude extracts of potato tubers and spinach
leaves ................ ........ ............ ... .... 29

2-3 Effect of P-mercaptoethanol on the extraction of
DS-Mn in potato tubers and spinach leaves.......... 30

2-4 Effects of aromatic amino acids and intermediary
metabolites on spinach DS-Mn at different pH's..... 31

3-1 Thermostability of spinach DS-Co .................... 48

3-2 Relative velocity of spinach DS-Co with various
substrates......................................... 54

3-3 Kinetic parameters for various substrates of spinach
DS-Co......... ............ ..... ............... 58

3-4 Relative configurations of hydroxy groups at C4 and
C5 of enzymatic products of spinach DS-Co with
various substrates as inferred from periodate
ox dation kinetics................................. 59

4-1 Thermostability of spinach KDOP synthase........... 74

4-2 General presence of KDOP snythase in higher
plants ......................... ........... ...... 75













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


ISOLATION AND CHARACTERIZATION OF KDOP SYNTHASE AND TWO
ISOZYMES OF DAHP SYNTHASE IN Spinacia oleracea L. AND
Solanum tuberosum L.


by

RON LOU DOONG

December 1990


Chairman: Roy A. Jensen
Major Department: Microbiology and Cell Science

Three enzymes, capable of condensing PEP and E4P to form

DAHP, were separated by DEAE-cellulose chromatography and

identified as DAHP synthase-Mn (DS-Mn), DAHP synthase-Co (DS-

Co), and KDOP synthase, respectively.

The plastidial isozyme, DS-Mn, was highly specific for

both substrates, i.e., PEP could not be replaced by pyruvate,

and E4P could not be substituted by glyceraldehyde 3-phosphate

or glycolaldehyde. It was a hysteretic enzyme, and a lag was

observed in standard assay conditions. The enzyme required DTT

for activity, and could be further stimulated by Mn". EDTA at

0.05 mM concentration completely inhibited its activity. The

optimum pH was 8.0, and the optimum temperature was 490C for

its activity. Activity was strongly inhibited by 0.7 mM


vii







arogenate at pH 7.0. Thioredoxin could stimulate its activity

only in the presence of DTT, suggesting that this enzyme is

tightly regulated by light as are many other chloroplast

enzymes involved in photosynthetic carbon assimilation. Its

rapid induction by mechanical wounding indicates that the

enzyme may be involved in the defense mechanism triggered by

this stress condition.

The cytosolic isozyme, DS-Co, on the other hand, used an

array of substrates with carbon length ranging from 2 to 4,

glycolaldehyde being the best substrate tested based on its

high specificity constant (Vmax/Km). It required divalent

metals for activity. At equimolar concentration of 0.5 mM, Co*+

was the best, Mn* the second, and Mg* the third. The pH

optimum was 9.5 and temperature optimum for activity was 490C.

The regulation of this enzyme has not been established, and a

new role in plant metabolism is being investigated.

Owing to its substrate ambiguity, KDOP synthase was

identified for the first time in higher plants. It possessed

weak activity as 3-deoxy-D-arabino-heptulosonate-7-phosphate

(DAHP) synthase. In the presence of phosphoenolpyruvate, which

conferred dramatic thermostability, KDOP synthase had a

catalytic temperature optimum of about 53C. The pH optimum

was 6.2, and divalent cations were neither stimulatory nor

required for activity. The Km values for arabinose-5-P and

phosphoenolpyruvate were 0.27 mM and about 35 jM,

respectively. The kinetics of periodate oxidation of KDOP


viii







formed by spinach KDOP synthase indicate that the same

stereochemical configuration exists as with bacterial KDOP.













CHAPTER I
LITERATURE REVIEW AND RATIONALE


Since the initial investigation on the formation of 3-

deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) in sweet

potato roots by Minamikawa and Uritani (1967), the number of

isozymes and regulation of DAHP synthase in algae and higher

plants has been a subject of controversy. Only one form of the

enzyme had been reported in mung bean shoots (Minamikawa,

1967), Euglena aracilis (Weber and Bock, 1968), cauliflower

florets (Huisman and Kosuge, 1974) before three isozymes with

different pH optima were demonstrated in the cotyledons of

Pisum sativum by the use of ammonium sulfate gradient

solubilization in 1976 (Rothe et al.), although the regulation

of the three isozymes was not investigated. In 1980 Graziana

and Boudet reported that the extraction of the enzyme in the

shoots of Zea mays required the presence of thiol compounds

such as dithiothreitol or P-mercaptoethanol. The activity of

the enzyme was inhibited in vitro by tryptophan. The enzyme

was retarded on a tryptophan agarose affinity column, and it

could be eluted with buffer containing tryptophan. Inhibition

of the enzyme was pH dependent and the magnitude of inhibition

increased during development. The sensitivity of the enzyme to

tryptophan was rapidly lost even when stored at low







2

temperature. The specific activity of the enzyme from roots

and shoots decreased with the development of corn seedlings.

The enzyme from extracts of roots was found to be insensitive

to tryptophan at any stage of the development. Isozymes could

not be demonstrated in this monocot, yet this was the first

report of a regulated DAHP synthase in higher plants.

Inhibition of the enzyme by tyrosine was first reported by

Byng et al. (1981) in Euglena gracilis, inhibition being

noncompetitive with respect to either phosphoenolpyruvate or

erythrose 4-phosphate. Reinink and Borstlap (1982) reported

that the enzyme activity in the crude extracts from pea leaves

was strongly inhibited by tyrosine and slightly stimulated by

phenylalanine and tryptophan; the inhibition could also be

relieved by the latter two aromatic amino acids. They

concluded that about 85% of the enzyme activity was

inhibitable and the remaining insensitive activity might be

due to partial desensitization of the enzyme during the

ammonium sulfate fractionation. In contrast, the enzyme in

extracts from Daucus carota suspension-cultured cells was

reported to be activated by tyrosine and tryptophan, the

activation being dependent upon the time after transfer of the

cells to fresh media(Suzich et al., 1984). The number of the

isozymes from carrrot roots was not studied (Suzich et al.,

1984) until 1985 when three activities were separated by

chromatography on phosphocellulose (Suzich et al., 1985).

Enzyme III was hysteretic and could be activated by







3

physiological concentrations of tryptophan. The three isozymes

also shared antigenic determinants.

Two differentially regulated isozymes of DAHP synthase

with very different pH optima and regulatory properties,

designated DS-Mn and DS-Co, were separated from seedlings of

Vigna radiata [L.] Wilczek by DEAE-cellulose column

chromatography (Rubin and Jensen, 1985). The activity of DS-Mn

was activated by chorismate, and was inhibited by prephenate,

L-arogenate, and tryptophan while DS-Co was not sensitive to

allosteric control. The inhibition of DS-Mn by tryptophan was

shown to increase with decreasing pH. This two-isozyme system

was further demonstrated in extracts from suspension-cultured

cells of Nicotiana silvestris and leaves of tobacco (Ganson et

al., 1986). DS-Mn required dithiothreitol for activity and was

stimulated by manganese, while DS-Co required divalent metals

for activity. Due to their diametrically different properties

in the requirements of DTT and divalent metals for activation,

pH optima, and substrate saturation concentrations with

respect to erythrose-4-phosphate, selective assays are

possible for the detection of either isozyme in a mixture of

the two. DS-Mn was demonstrated to be localized in the

chloroplast; whereas DS-Co was thought to be in the cytosol

for there was little, if any, activity in the chloroplast. The

two isozymes were shown to respond differently to transfer of

the suspension-cultured cell of M. silvestris to fresh media;

DS-Mn activity declined substantially in stationary phase and







4

reached its peak in early exponential phase; whereas DS-Co

activity peaked in late exponential phase. In contrast, only

one enzyme, similar to DS-Mn, was reported in the tubers of

Solanum tuberosum L. (Pinto et al., 1986) and a cDNA encoding

this enzyme has been cloned (Dyer et al., 1990). Glyphosate

was shown to induce the activity of this DAHP synthase in

suspension-cultured cells of potato (Pinto et al., 1988)

although it had no effect on the enzyme activity in vitro. A

glyphosate-tolerant tobacco cell line, N. tabacum L. Indiana

(17), was later selected from the glyphosate-sensitive line

and demonstrated to have elevated DAHP synthase activity (Dyer

et al., 1988).

Since potato and tobacco belong to the family of

Solanaceae, it seems surprising that the biochemistry of these

two plants would differ so much as to have one isozyme on the

one hand and two isozymes on the other. Although DAHP

synthases in spinach (Spinacia oleracia L.) have been

investigated with crude extracts and extracts of purified

chloroplast to establish their subcellular location,

chromatographic separation has not yet been achieved. The

objectives of this investigation are to establish the number

of isozymes of DAHP synthase by DEAE-cellulose column

chromatography in g. tuberosum and S. oleracia as they

represent nonphotosynthetic and photosynthetic tissues,

respectively in higher plants, and to further characterize the

enzymes obtained from chromatography with respect to their







5

substrate specificities, pH and temperature optima, metal and

thiol compound requirements, and regulation.











CHAPTER II
DAHP SYNTHASE-Mn (DS-Mn)


Introduction

DAHP synthase-Mn (DS-Mn) is the plastidial enzyme in

higher plants for the committed step of aromatic amino acid

biosynthesis. It has been partially purified and characterized

in mung bean (Rubin and Jensen, 1985), tobacco suspension-

cultured cells (Ganson et al., 1986) by DEAE-cellulose

chromatography, and from spinach by chloroplast purification

(Ganson et al., 1986). This enzyme is undoubtedly the one that

catalyzes the condensation of PEP and E4P to form DAHP as the

precursor for the synthesis of phenylalanine, tyrosine, and

tryptophan in the chloroplast. The DAHP synthases documented

in literature could be either DS-Mn or DS-Co on the basis of

substrate ambiguity that was observed with DS-Co, requirement

of reducing agents, or divalent cations for catalysis. Hence,

the first DAHP synthase reported in mung bean shoots, by

Minamikawa (1967) seemed to be DS-Mn since the enzyme showed

activity without divalent cations. The purified enzyme from

cauliflower florets (Huisman and Kosuge, 1974) also behaved

like DS-Mn for glyceraldehyde-3-phosphate or glyceraldehyde

was not used as substrates. The DAHP synthase in corn

(Graziana and Boudet, 1980) was probably the first DS-Mn type

enzyme ever reported in monocots for its requirement of thiol

6






7

compounds in the extraction of the active enzyme, and for its

activity without divalent cations. However, all of these

investigations were made with plant tissues that are not

photosynthetically active. The first report of DAHP synthase

in higher plant tissues with vigorous photosynthetic activity

was from pea leaves (Reinink and Borstlap, 1982); however, the

enzyme was only partly purified by ammonium sulfate

fractination. DS-Mn in spinach leaves was detected in isolated

chloroplast, yet simultaneous demonstration of the two

isozymes by an ion-exchanger chromatography was never

attempted. This chapter describes the response of the two

isozymes to mechanical wounding, a fast and yet complete

separation of DS-Mn from DS-Co and KDOP synthase in potato

tubers and spinach leaves, its enzymological properties, and

its regulation by intermediary metabolites of aromatic amino

acid biosynthesis.



Materials and Methods

Plant Material

Spinach and Idaho potatoes were purchased from a local

supermarket, washed with deionized water, frozen with liquid

nitrogen, and ground to a fine powder by use of a Waring

blender. The powders were stored at -700C prior to extract

preparation.









Plant Extract Preparation

All procedures were carried out at 0-40C. A 45-g amount

of powder was mixed with 30 ml of buffer A (50 mM K phosphate,

pH 7.2, containing 0.5% P-mercaptoethanol) and thawed at room

temperature. The extract was clarified by centrifugation at

29,000 x g for 30 min and filtered through Miracloth. A one-

tenth volume of 2% protamine sulfate in buffer A was slowly

added to the extract and stirred for 10 min. The precipitate

was removed by centrifugation at 29,000 x g for 20 min.

Spinach and potato extracts used for column

chromatography were further treated as follows. The foregoing

supernatants were brought to 70% (spinach) or 60% (potato) of

saturation with finely ground ammonium sulfate and stirred for

10 min. The protein precipitate was collected by

centrifugation at 29,000 x g for 20 min, and resuspended in a

minimal volume of buffer B (10 mM EPPS, pH 7.5, and 50 mM

KC1). Desalting was accomplished by passage through Sephadex

G-25 (PD-10) columns equilibrated with buffer B according to

the manufacturer's instructions.

DE-52 Column Chromatography of Plant Extracts and Enzyme
Assays

A 150-mg (84-mg for potato) amount of protein was loaded

onto a DE-52 anion exchanger column (1.5 x 19 cm) equlibrated

with buffer B. The column was washed with 3 bed volumes of the

buffer before a 400 ml gradient (50-300 mM KC1) in buffer B

was applied. For the potato extracts a 400 ml gradient (50-500






9

mM KC1) was employed. The flow rate was 30 ml/hr, and

fractions of 2.9 ml were collected.

Enzyme Assays

Assays were executed at 37C for 20 min unless otherwise

indicated in 200 Al reaction mixtures containing 50 mM EPPS

(pH 8.0), 12.5 mM KC1, 3 mM PEP, 0.6 mM E4P, 0.5 mM DTT and

0.5 mM MnC12 for DS-Mn; 50 mM EPPS (pH 8.6), 40 mM KC1, 3 mM

PEP, 3 mM E4P, and 10 mM MgC12 for DS-Co; 50 mM BTP (pH 6.5),

3 mM PEP, and 3 mM arabinose-5-P for KDOP synthase. The

reaction was stopped with 50 pl TCA (20%) and the enzymatic

products were assayed as follows.

Chemical Assays

DAHP was assayed as described by Jensen and Nester

(1966), using the chemical method of Weissbach and Hurwitz

(1959) as adapted by Srinivasan and Sprinson (1959). The

absorbance at 549 nm was measured in a thermostatically

controlled auto-sampler cuvette set at 550C.

Protein Assays

Protein concentrations were determined by the method of

Bradford (1976) using BSA as a standard.

Mechanical Wounding of Potatoes

Tubers of Idaho potatoes were purchased from a local

supermarket, washed with deionized water, and soaked in a

streptomycin sulfate solution (25 pg/ml). Disks of 12 mm

diameter by 13 mm thickness were excised from the center

tissue of the tubers and placed in petri dishes (15 cm







10

diameter) containing Whatman #3 filter paper saturated with

streptomycin sulfate solution. The petri dishes were wrapped

in aluminum foil and stored in a cabinet at room temperature.

Disks harvested at zero time, 12, 24, 35, 48, 72, 96, 120, and

144 h, were frozen in liquid nitrogen and ground to a fine

powder in a Waring blender. Powders were stored at -800C

before use.

Biochemicals

PEP (monocyclohexylammonium salt), glycolaldehyde, D-

glyceraldehyde, L-glyceraldehyde, DL-glyceraldehyde-3-

phosphate, D-erythrose, L-erythrose, D-threose, L-threose, D-

erythrose-4-phosphate, protamine sulfate, and buffers

(EPPS,BTP) were obtained from Sigma. DTT was purchased from

Research Organics, (Cleveland, OH). Thioredoxin of E. coli was

from CalBiochem, (La Jolla, CA). DE-52 anion exchanger was

obtained from Whatman, Inc. (Clifton, NJ). PD-10 columns were

from Pharmacia (Piscataway, NJ).



Results

Construction of Standard Curve of DAHP Using Authentic KDO as
a Standard

The extinction coefficient was calculated to be 85,500

M'Icm'' using authentic KDO as a standard, and this value was

used throughout this dissertation.

Separation of DS-Mn from DS-Co by DE-52 Chromatographv

Since the liability of DS-Mn from tobacco suspension-

cultured cells was well appreciated, an attempt was made to







11

achieve a fast and complete separation of two isozymes of DAHP

synthase with DEAE-cellulose chromatography. A buffer

containing 10 mM EPPS at pH 7.5 and 50 mM KC1 proved to serve

this purpose. Under this condition DS-Mn eluted in the wash

while DS-Co was retarded by the anion exchanger. A third

enzyme, that proved to be KDOP synthase (see chapter IV), was

also identified by virture of its ability to utilize E4P as a

cosubstrate (Fig. 2-1 and 2-2).

Thermostability

The enzyme was very labile even at room temperature in

the absence of PEP. It lost about 10% of the activity as

compared with the enzyme in the presence of PEP at 240C for 30

min. At 37C only 46% activity could be recovered when PEP was

present; whereas 32% activity remained when PEP was absent

during thermal treatment (Table 2-1).

pH and Temperature Optima

The reaction velocity of DS-Mn from spinach rose with pH

up to 8.0, then dropped sharply with the pH higher than

8.0 (Fig 2-3). This was also true for DS-Mn from potato tuber.

Temperature optimum was 49C, similar to that of DS-Co (Fig.

2-4).










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Fig. 2-2. Elution profiles of spinach DS-Mn, DS-Co, and
KDOP synthase from DEAE cellulose. A 150-mg amount of spinach
extract prepared as described in Materials and Methods was
applied. The salt gradient was 50 to 300 mM KC1 in buffer B.
The A.4 values plotted on the ordinate scale were obtained by
incubation of 50 Al aliquots of enzyme for 20 min at 37C.










































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TABLE 2-1. Thermostability of spinach DS-Mn


% of control activity

Thermal treatment + PEP PEP

240C 100 89

37oC 46 32

470C 7 5

570C 0 0


Partially purified enzyme was incubated with or without
1.1 mM PEP at the indicated temperature for 30 min and removed
to an ice bath prior to assay at 37C under standard
conditions. 100% activity is equivalent of 19.8 nmol.


Substrate Specificity

DS-Mn exhibited an absolute specificity for both PEP and

E4P. Thus, PEP could not be replaced by pyruvate, and E4P

could not be replaced by any of the alternative sugar

substrates such as glycolaldehyde, D-glyceraldehyde, L-

glyceraldehyde, DL-glyceraldehyde-3-phosphate, D-erythrose, L-

erythrose, D-threose, or L-threose.

Divalent Metal Effects

Unlike DS-Co, DS-Mn from spinach did not require divalent

cations for activity; however, at optimal concentration of 0.5

mM, its activity could be further enhanced 47% and 9% by MnC12

and MgCl2, respectively (Fig. 2-5). At the same concentration

of CoC12, inhibition of 60% was observed. EDTA at the

concentration of 0.05 mM completely inhibited the activity.







17

Dialysis of the enzyme treated with 1 mM EDTA against buffer

containing DTT only restored 15% of the activity when assayed

without divalent metal; however, upon the addition of 0.5 mM

MnCl2 or CaC12, the activity was restored 90% and 52%,

respectively, although CaCI2 did not stimulate the non-EDTA

treated enzyme activity. This indicated that the enzyme might

be a metalloprotein whose divalent cation could be readily

chelated by EDTA. This plastidial enzyme differs from DS-Co,

the cytosolic isozyme, in that the latter requires divalent

metals for activity.











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Effects of Dithiothreitol and Thioredoxin

The enzyme required dithiothreitol for activity. In the

presence of DTT, activity was increased two-fold by 25 Ag of

bacterial thioredoxin with partially purified enzyme. In the

absence of DTT, thioredoxin did not have any effect on the

enzyme (Fig. 2-6). The hysteretic lag of the progress curve

could be removed by pre-assay incubation of the enzyme with

PEP and DTT (Fig. 2-7). This is similar to many enzymes in

chloroplasts that are regulated by light. These redox

properties of DS-Mn were not observed for the cytosolic

isozyme, DS-Co. The stimulation of the enzyme in the crude

extracts of spinach by thioredoxin was not so remarkable as

that observed with crude extracts of potato (Table 2-2).

Perhaps the spinach extracts are relatively rich in endogenous

reduced thioredoxin compared to nonphotosynthetic potato

tubers.



Substrate Saturation Curves

Spinach DS-Mn showed sigmoid substrate saturation curves

with both E4P and PEP. E4P exhibited substrate inhibition at

concentrations higher than 0.6 mM. The enzyme was saturated by

3 mM PEP (Fig. 2-8 and 2-9).












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Fig. 2-6. Effects of DTT and thioredoxin on spinach DS-
Mn. Reaction mixtures contained 3 mM PEP, 0.6 mM E4P, 0.5 mM
MnCI2, 12.5 mM KC1, 50 mM EPPS at pH 8.0. DTT and thioredoxin
was 0.5 mM and 12.5 Mg, respectively, if present. The reaction
was followed for 30 min. The progress curve with thioredoxin
in the absence of DTT superimposes with the curve obtained
without either.


















































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29

TABLE 2-2. Effect of DTT and/or thioredoxin on DS-Mn
activity from crude extracts of potato tubers and spinach
leaves.


A549


Assay conditions Potato Spinach

No Addition 0.058 0.328

DTT (0.5 mM) 1.38 0.681

Thioredoxin (12.5 pg) 0.081 0.349

DTT + Thioredoxin 1.864 0.745



Extracts were made with 50 mM K-Phosphate buffer at pH
7.2 and 0.1% P-mercaptoethanol. Clarified extracts were
desalted into 10 mM EPPS buffer pH 8.0 and 50 mM KC1 by
passage through PD-10 columns.


Effect of 0-Mercaptoethanol on the Extraction of DS-Mn

Similar to the enzyme in tobacco suspension-cultured

cells, DS-Mn in potato tubers could only be detected when P-

mercaptoethanol was present in the extraction buffer. It

differed from DS-Mn in spinach leaves where the activity of

DS-Mn could be readily measured in the crude extracts prepared

without P-mercaptoethanol (Table 2-3). This might be due to

the presence of reduced thioredoxin in spinach leaf extracts

and its absence in potato tubers. Potatoes are rich in

phenolic compounds that may contribute to inactivate the

enzyme in the absence of reducing agents.







30

TABLE 2-3. Effect of p-mercaptoethanol on the extraction
of DS-Mn in potato tubers and spinach leaves


Assay Conditions Extraction Condition
No BME 0.5% BME

potato spinach potato spinach

No thiol reagent 0.027 0.358 0.186 1.053

DTT (mM) 0.125 0.021 0.793 0.846 1.209

0.25 0.017 0.875 1.077 1.145

0.5 0.013 0.903 1.30 1.055

1.0 0.009 0.852 1.235 0.904



Crude extracts were made with 50 mM KP buffer, pH 7.2,
with or without 0.5% P-mercaptoehtanol. The clarified extracts
were desalted into 10 mM EPPS buffer, pH 8.0, 50 mM KCl with
PD-10 columns. A49 was recorded after a 20 min duration of
reaction.


Effects of Aromatic Amino Acids and Their Intermediate
Metabolites on Spinach DS-Mn at Various pH's

DS-Mn was found to be sensitive to arogenate. The

inhibition was a function of pH; at pH 7.0, 65% of the

activity was inhibited, while at pH 8.0 only 23% of the

activity was inhibited. The concentration for 50% inhibition

was estimated to be 0.5 mM at pH 7.0 (Fig. 2-10). The

inhibition by arogenate was competitive and noncompetitive

with respect to E4P and PEP, respectively. A slight activation

of 10% was observed when the pH was raised to 9.0.

Phenylalanine, tyrosine, tryptophan, chorismate, prephenate,









and caffeic acid did not have much effect on the enzyme (Table

2-4).



TABLE 2-4. Effects of aromatic amino acids and
intermediary metabolites on spinach DS-Mn at different pH's


% control activity



effectors pH 7.0 pH 8.0 pH 9.0

PHE 109 99 91

TYR 110 94 102

TRP 97 98 100

CHA 95 108 110

PPA 110 105 100

AGN 35 77 110



AGN was used at 0.7 mM, all others were 0.5 mM. Reaction
tubes where respective effectors were added after enzyme
reaction was stopped with TCA served as controls. PHE, TYR,
TRP, CHA, PPA, and AGN stand for phenylalanine, tyrosine,
tryptophan, chorismate, prephenate, and arogenate,
respectively.


Induction of DS-Mn after Mechanical Wounding of Potato
Tubers

DS-Mn responded to mechanical wounding more rapidly and

in greater magnitude than DS-Co. Within 48 hours after

wounding treatment, the specific activity of DS-Mn increased

about seven-fold, while DS-Co responded slowly with only a

15% increase in the same period (Fig. 2-11). Mechanical







32

wounding provides a means of enriching DS-Mn in vivo for the

purpose of enzyme purification.


Selective Assays

The finding that DS-Co utilizes glycolaldehyde and G3P

facilitates a selective assay for DS-Co in the presence of

DS-Mn. Hence, when assayed with E4P under selective assay

conditions designed for DS-Co, DS-Mn showed 14% of its

activities in the disguise of DS-Co; when assayed with

glycolaldehyde or G3P as cosubstrates for DS-Co, DS-Mn

activity was completely discriminated. This provides a

convenient method to confirm if DS-Mn preparation is

contaminated with DS-Co in the process of purification.











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41

Discussion

DS-Mn is responsible for the first enzymatic step in the

biosynthesis of aromatic amino acids in the chloroplast. To

resolve the confusion over the number of isozymes in higher

plants in general and potato tubers in particular, results of

mechanical wounding on potato tubers lend a strong support to

the occurrence of a second isozyme, i.e., DS-Co. Complete

chromatographic separation of two activities by DEAE cellulose

further provided solid evidence for dual pathway hypothesis.

DS-Mn has been shown to elevate rapidly in suspension-cultured

cells of N. silvestris in response to subculture (Ganson and

Jensen, 1987). Wounding might trigger what is in essence a

growth response (Morris et al., 1988). In fact, wounding was

shown to induce mRNA encoding DAHP synthase followed by the

increased synthesis of the enzyme in potato or tomato tissue,

and a similar effect was also observed with phenylalanine

ammonia-lyase (Dyer et al., 1989). Induction of DS-Mn, not DS-

Co, in cultured parsley cells by a cell wall fraction of the

fungus Phvtophthora megasperma was also reported (McCue and

Conn, 1989). The nonresponsiveness of the enzyme to mechanical

wounding and its activity without DTT reported in sweet potato

roots by Minamikawa and Uritani (1966) indicated that the

enzyme activity detected was obviously not that of DS-Mn. It

is intriguing that DS-Mn responded much faster than its

cytosolic counterpart while most, if not all, enzymes of

secondary metabolism function in the cytosol (Jensen, 1986).








There might be a rapid export of phenylalanine into the

cytosol after the induced biosynthesis of this precursor for

secondary metabolism.

The requirement of DTT for activation and further

enhancement by bacterial thioredoxin suggested that DS-Mn,

like many chloroplast enzymes involved in the photosynthetic

carbon assimilation, is also tightly regulated by light

(Buchanan, 1980; Crawford et al., 1986; Jacquot et al., 1984;

Jensen, 1986). Two forms of thioredoxin have been reported in

the chloroplast of spinach i.e., thioredoxin f and

thioredoxin m (Wolosiuk et al., 1979). The question of which

one is capable of stimulating DS-Mn activity merits further

investigation. Stimulation by thioredoxin of shikimate kinase

from spinach chloroplast has also been reported (Schmidt and

Schultz, 1987), but the effect was later shown to be

stabilization rather than activation (Schmidt et al., 1990).

Induction of phenylalanine ammonia-lyase by light in potato

tuber disks (Zucker, 1968; Sacher et al., 1972) paralleled by

the increase of chlorogenic acid (Zucker, 1965) have also been

demonstrated. In view of the parallel response of DS-Mn and

PAL to mechanical wounding, stimulation by reduced

thioredoxin, induction by the light, these two enzymes seem to

play a concerted role in the synthesis of secondary

metabolites such as phenylpropanoids and phytoalexins as a

defense mechanism.







43

Both inhibition and activation of DAHP synthase by

aromatic amino acids have been reported in higher plants

although in most cases insensitivity was observed. A pH

dependent inhibition of the enzyme in corn was also reported

with respect to tryptophan in corn (Grazinia and Boudet, 1980)

and in seedlings of Vigna radiata (Rubin and Jensen, 1985). In

both cases the magnitude of inhibition increased with

decreasing pH. On the other hand, tryptophan was reported to

activate the enzyme from carrot roots (Suzich et al., 1985)

and potato tubers (Pinto et al., 1986). Tyrosine was shown to

be inhibitory for the enzyme from pea leaves (Reinink and

Borstlap, 1982), but stimulatory for the enzyme from

suspension-culture cells of carrot (Suzich et al., 1984). In

this investigation, DS-Mn from spinach leaves could be

demonstrated to be inhibited only by arogenate, and the

inhibition was pH dependent, similar to the inhibition of

chorismate mutase I (the chloroplast isozyme in the aromatic

amino acid biosynthetic pathway) by tyrosine and phenylalanine

(Goers and Jensen, 1984). The greater inhibition at pH 7.0

than at pH 8.0 suggests that in the light the enzyme is more

active than in the dark. This pattern of regulation is

consistent with a sequential model of feedback inhibition of

aromatic amino acid biosynthesis in the chloroplast (Gaines et

al., 1982; Jensen, 1985; Jung et al., 1986).












CHAPTER III
DAHP SYNTHASE-Co (DS-Co)


Introduction

Since two isozymes of DAHP synthase were established in

higher plants such as mung beans, tobacco, spinach, and

potatoes, the in vivo function of the cytosolic form of the

isozyme DS-Co has not been established. In this investigation,

the enzyme from spinach leaves and potato tubers

(representative of photosynthetic and nonphotosynthetic

tissues, respectively) was completely separated from DS-Mn by

DEAE-cellulose column chromatography and has been further

characterized in comparison with its chloroplast counterpart.

Substrate specificity of the enzyme was extensively

investigated as a potential role of the enzyme in a new

biochemical pathway was suspected.


Materials and Methods

Plant Material

Spinach and Idaho potatoes were purchased from a local

supermarket. All plant material was washed with deionized

water, frozen with liquid nitrogen, and ground to a fine

powder by use of a Waring blender. The powders were stored at

-70C prior to extract preparation.

44









Plant Extract Preparation

All procedures were carried out at 0-4C. A 45 g amount

of powder was mixed with 30 ml of buffer A (50 mM K phosphate,

pH 7.2, containing 0.5% B-mercaptoethanol) and thawed at room

temperature. The extract was clarified by centrifugation at

29,000 x g for 30 min and filtered through Miracloth. A one-

tenth volume of 2% protamine sulfate in buffer A was slowly

added to the extract and stirred for 10 min. The precipitate

was removed by centrifugation at 29,000 x g for 20 min.

Spinach and potato extracts used for column

chromatography were further treated as follows. The foregoing

supernatants were brought to 70% (spinach) or to 60% (potato)

of saturation with finely ground ammonium sulfate and stirred

for 10 min. The protein precipitate was collected by

centrifugation at 29,000 x g for 20 min and resuspended in a

minimal volume of buffer B (10 mM EPPS, pH 7.5, and 50 mM

KC1). Desalting was accomplished by passage through Sephadex

G-25 (PD-10) columns equilibrated with buffer B according to

the manufacturer's instructions.

DE-52 Column Chromatographv of Plant Extracts and Enzyme
Assays

150 mg spinach protein (or 84 mg potato protein) was

loaded onto a DEAE-cellulose column (1.5 x 19 cm) equilibrated

with buffer B. The column was washed with 3 bed volumes of

buffer before a 400 ml gradient (50-300 mM KC1) in buffer B

was applied. For the potato extracts a 400 ml gradient (50-500

mM KC1) was employed. The flow rate was 30 ml/hr, and







46

fractions of 2.9 ml were collected. The enzyme assays were

carried out as described previously in Chapter II unless

otherwise stated.

Chemical Assays

The analytical assays were carried out as described in

Chapter II except that periodate oxidation was 10 min for the

enzymatic products with glyceraldehyde 3-phosphate, 20 min for

the products with glycolaldehyde, and 30 min for the products

with other sugars.

Biochemicals

PEP (monocyclohexylammonium salt), glycolaldehyde,

glyoxylate, D-glyceraldehyde, L-glyceraldehyde, DL-

glyceraldehyde 3-phosphate, D-erythrose, L-erythrose, D-

threose, L-threose, D-erythrose-4-phosphate, D-ribose, L-

ribose, D-arabinose, L-arabinose, D-xylose, L-xylose, D-

lyxose, L-lyxose, D-ribose-5-phosphate, D-arabinose-5-

phosphate, D-glucose-6-phosphate, protamine sulfate, and

buffers (EPPS, BTP, and CHES) were obtained from Sigma. DTT

was purchased from Research Organics, (Cleveland, OH), and DE-

52 Anion exchanger was obtained from Whatman, Inc.(Clifton,

NJ). PD-10 columns were from Pharmacia (Piscataway, NJ).


Results

Separation of DS-Co from DS-Mn and KDOP Synthase by DE-52
Anion Exchanger Chromatoaraphv

DS-Co from spinach leaves and potato tubers both eluted

from the anion exchanger at about 0.15 M KC1 (Fig. 2-1 and 2-







47

2). A minor peak preceding the peak activity of DS-Co was

detected in both cases under the assay condition for DS-Co.

Unlike DS-Mn and DS-Co, this new activity did not require DTT

or metal, which were required for DS-Mn or DS-Co respectively,

for activation (Morris et al., 1989). It was later confirmed

to be a new enzyme, i.e., KDOP synthase (see Chapter IV of

this dissertation).

Thermostabilitv

The enzyme was unstable in the absence of PEP; it lost

more than 90% of activity after incubation at 370C for 30 min.

However, in the presence of PEP, 90% of the activity remained

(Table 3-1).

pH and Temperature ODtima

The enzyme was almost inactive at neutral pH. Activity

rose with the pH and reached its peak at pH around 9.5. This

could further facilitate the fine-tuning of selective assay

for DS-Co in the presence of DS-Mn. The enzyme had a

temperature optimum at 49C, but was rapidly inactivated at

higher temperatures (Fig. 2-3 and 2-4).

Saturation Curves of the Enzyme by PEP with Various
Cosubstrates

DS-Co was saturated by 1.5 mM PEP when assayed with 3 mM

E4P, 6mM G3P, or 8 mM glycolaldehyde. All the curves were

sigmoid.







48

TABLE 3-1. Thermostability of spinach DS-Co


% of control activity

Thermal treatment + PEP PEP

27C 100 86

370C 90 5

47C 4 1

57C 0 0



Partially purified enzyme was incubated with or without
1.1 mM PEP at the indicated temperature for 30 min and removed
to an ice bath prior to assay at 37C under standard
conditions.


Divalent Metal Requirement

Like the enzyme from tobacco, DS-Co from spinach or

potatoes exhibited no activity without divalent metal. At the

equimolar concentration of 0.5 mM, Cod* was the best, Mn* the

second, and Mg* the third with relative velocity of 100,

16, and 10, respectively. Mn++ and Co++ became inhibitory as

the concentrations went higher than 1 mM, while Mg* showed its

maximal activation at 20 mM. Hence, at their maximal

activation concentration (20 mM for Mg+ and 1 mM for Co" and

Mni), the relative velocity was 100, 70, and 15, respectively

(Fig. 3-1).









Substrate Ambiguity

DS-Co from spinach and potato had the same properties

with respect to substrate specificity, i.e., they both

utilized an array of sugars ranging from carbon length of 2 to

4 to make the corresponding 2-keto-3-deoxy sugar acids, as

shown in Table 3-2. Glycolaldehyde exhibited the highest

reaction velocity when all sugars were used at 3mN

concentrations. Pentoses were poor substrates except when

phosphorylated, an effect due to the stabilization by the

phosphate group of a significantly greater percentage of the

open-chain form of pentose phosphates such as D-ribose 5-

phosphate and D-arabinose 5-phosphate. No discrimination

between the steroisomers was observed as comparable velocities

were demonstrated with either D or L isomer of glyceraldehyde,

erythrose, or threose. Surprisingly, glyoxylate was also

usable in this enzymatic reaction. The enzyme was obviously

not a reversible aldolase since pyruvate failed to substitute

for PEP.

On the basis of Michaelis constant, D-erythrose 4-

phosphate seemed to be the best substrate because it had a Km

value of 1.95 mM. However, glycolaldehyde had the largest Vmax

value and a largest Vmax/Km value although it had a very large

Km value compared with erythrose 4-phosphate (Table 3-3).









ta t

0-I





oo
9.,4


*.~0 90
0 0
000
*r"


4)- .1-I 4-)
4 4
14U1



rl) 4o

o L





-r4 rU4


H r4




> C) O4




I qjH
UU '(


( r (













0 M
o 0 0

04- 0

4) tDY

4Z1 t- 4


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W 0O




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t; w r

-r
pa 9
04 H





















I-e

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0 J

o a o







0 en ci 4













A AIIIA IOYIIX& %
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A1IA11OV -IVWIXVW %





























Fig. 3-2. Saturation curves of spinach DS-Co by PEP with
various cosubstrates. The reaction mixture contained 10 mM
MgC12, 40 mM KC1, 50 mM EPPS at pH 8.6, 3 mM E4P, (or 6 mM DL-
G3P, or 8 mM glycolaldehyde), and PEP at indicated
concentrations.



































0
E
B I A






0.5-
0



0 0.5 1.0 1.5 2.0 2.5 3.0

mM PEP









TABLE 3-2.
various substrates


54

Relative velocity of spinach DS-Co with


Substrates

glycolaldehyde

D-glyceraldehyde

L-glyceraldehyde

DL-glyceraldehyde-3-phosphate

D-erythrose

L-erythrose

D-threose

L-threose

D-erythrose-4-phosphate

D-ribose

L-ribose

D-arabinose

L-arabinose

D-xylose

L-xylose

D-lyxose

L-lyxose

D-ribose-5-phosphate

D-arabinose-5-phosphate

D-glucose-6-phosphate

glyoxylate


Relative Velocity

245

176

212

142

93

70

92

52

100 (1.98 nmol/min)

1

1

0.7

0

1

1

1.8

1.8

12

8

0.5

20


All the substrates above were
concentrations for standard assays.


present at 3 mM


j







55

Confirmation of Reaction Products

The reaction products all had an absorption peak at 549

nm by periodate-thiobarbituric acid procedure, indicating the

presence of 3-deoxy aldulosonic acid similar to DAHP

molecules. This has been further confirmed by Malcolm O'Neill

of the Complex Carbohydrate Research Center in Athens,

Georgia. However, the relative configuration of hydroxy group

at the fourth and fifth carbons has not been elucidated yet.

From the kinetics of periodate oxidation (Fig. 3-3), all

products seemed to have a trans configuration with respect to

these two carbons, except that derived from D-glyceraldehyde

3-phosphate, which formed a chromogen rapidly destroyed in the

oxidation procedure (Table 3-4).





























Fig. 3-3. Kinetics of periodate oxidation of enzymatic
products of spinach DS-Co with various substrates.






















,DL-G3P lIvaInInlarahvut


D-E4P


I I I I I
10 20 30 40 50

PERIODATE OXIDATION TIME (min)


100-


80-



60-
40-



40-



20-







58

TABLE 3-3. Kinetic parameters for various substrates of
spinach DS-Co


Km (mM)

glycolaldehyde 8.6

glyoxylate 3.6

D-glyceraldehyde 3.5

L-glyceraldehyde 3.3

DL-glyceraldehyde- 3.0

3-phosphate

D-erythrose 2.5

L-erythrose 5.1

D-threose 8.4

L-threose 13.9

D-erythrose 4-phosphate 1.95


Vmax (nmol/min)

20

0.47

5.0

5.4

5.3



2.2

2.8

5.2

5.0

3.1


PEP at 3 mM was used in combination with
substrates under standard assay conditions.


Regulation

The enzyme was not affected by phenylalanine, tyrosine,

tryptophan, chorismate, prephenate, or arogenate when tested

at 0.5 mM. Thioredoxin was not stimulatory.


Vmax/Km

2.32

0.13

1.43

1.64

1.76



0.88

0.55

0.62

0.36

1.57


various







59

TABLE 3-4. Relative configurations of hydroxy groups at
C4 and C5 of enzymatic products of spinach DS-Co with various
substrates as inferred from periodate oxidation kinetics


Substrates

glycolaldehyde

D-glyceraldehyde

L-glyceraldehyde

D-erythrose

L-erythrose

D-threose

L-threose

DL-glyceraldehyde-3-phosphate

D-erythrose 4-phosphate


Configuration

trans

trans

trans

trans

trans

trans

trans

cis

trans


Discussion

Owing to its insensitivity to feedback inhibition by

three aromatic amino acids and their intermediary metabolites,

the role of DS-Co in aromatic amino acid biosynthesis has been

a subject of interest. The evidence that this enzyme utilized

many simple sugars other than E4P, the conventional substrate

for DAHP synthesis, shed further light on its possible role in

a hitherto unrecognized biochemical pathway. The possible

implications of the enzyme in plant metabolism are discussed

as follows.






60

Metabolism of Two Carbon Compounds

It is still unknown whether glycolaldehyde is an

intermediary metabolite in the biochemistry of plants. Since

the report of glycolaldehyde dehydrogenase in 1960 by Davies,

there has been no further investigation. Sources of

glycolaldehyde could be decarboxylation of hydroxypyruvate,

which in turn was derived from oxidation of glycerate or

phosphoglycerate, or from the transamination of serine. The

possibility that glycolaldehyde could be released from an

active glycolaldehyde-enzyme complex during transfer reaction

catalyzed by transketolase as reported in 1961 by Datta and

Racker could also be considered.

Although the biogenesis of glycolaldehyde in the plant

kingdom is still a mystery, 2-keto-3-deoxy-arabonate, the

enzymatic product of DS-Co, originally isolated and

characterized by Palleroni and Doudoroff in 1956, has been

identified as an intermediary metabolite for the catabolism of

arabinose to 2-ketoglutarate, and the following two catalytic

steps, i.e., dehydration followed by dehydrogenation, have

been documented in Pseudomonas saccharophila (Weimberg, 1959;

Stoolmiller and Abeles, 1966). The source of a-ketoglutarate

required for transport into chloroplasts for reassimilation of

ammonia released by photorespiration is unknown although its

origin has been proposed to be within the mitochondria.

Glyoxylate, on the other hand, could be utilized by DS-Co

to form a 4-hydroxy-2-oxoglutarate (4-HOG), which could also







61

generate a-ketoglutarate in two enzyme steps. A recent report

of an aldolase in E. coli which catalyzes the reversible

formation of 4-HOG from pyruvate and glyoxylate supports this

possibility (Patil and Dekker, 1990). Glyoxylate could be

produced in vivo by means of (a) oxidation of glycolate from

photorespiration (Ogren, 1984), (b) transamination of glycine

(Harder and Quayle, 1971), (c) glyoxylate cycle by the

activity of isocitrate lyase (Zelitch, 1988), and (d)

degration of allantoin (Thomas and Schrader, 1981). Hence,

there exists a potential pathway for the replenishment of a-

ketoglutarate via the condensation of PEP and either

glycolaldehyde or glyoxylate by DS-Co activity. These

reactions are potentially significant in vivo because they

could provide the cell with an alternative anaplerotic pathway

to a-ketoglutarate.

Metabolism of Three Carbon Compounds

The serendipitous discovery of D-glyceraldehyde 3-

phosphate as a good substrate for DS-Co in higher plants has

led to a hypothetical pathway where aromatic amino acid

biosynthesis in cytosol may originate with the condensation of

PEP with D-glyceraldehyde-3-P rather than D-erythrose-4-

phosphate. This hypothetical phyto-shikamate pathway utilizes

intermediates such as phyto-shikimate, phyto-chorismate, and

phyto-prephenate molecules which differ from their

conventional counterparts in replacement of the 1-carboxy

substituent by a 1-hydroxy substituent. According to this







62

scheme, it is fortuitous that the cytosolic DS-Co and

chorismate mutase-2 possess the substrate ambiguity to accept

erythrose-4-P phosphate and chorismate in place of

glyceraldehyde-3-P and phyto-chorismate, respectively. The

much greater availability of triosephosphate than of

erythrose-4-phosphate in the cytoplasm may accommodate the

quantitatively great transit of carbon through the aromatic

pathway to multiple connecting pathways of secondary

metabolism.

2-Keto-3-deoxy-6-phosphogluconic acid was originally

established as an intermediary metabolite of glucose

metabolism in F. saccharoDhila in 1954 by MacGee and Doudoroff

and its nonphosphorylated form, 2-keto-3-deoxygluconate, a

possible product of DS-Co with D-glyceraldehyde-3-phosphate

followed by dephosphorylation, has been reported to be a

degradation product of gluconate by Asperqillus nicer (Elzainy

et al., 1973). On the other hand, 2-keto-3-deoxygalactonate

and its phosphate ester were found to be a product of

galactose metabolism in Gluconobacter liauefaciens

(Stouthamer, 1961). Whether these groups of 2-keto-3-deoxy-

sugar acids are involved in the hexose metabolism of higher

plants has never been investigated.

Metabolism of Four Carbon Compounds

As a product of the oxidative pentose phosphate pathway,

erythrose-4-phosphate may be an additional source of aromatic

amino acid biosynthesis in the cytoplasm.







63

Structural Component of Phvtotoxin and Cell Wall

2-Keto-3-deoxygluconic acid, a possible enzymatic product

of DS-Co with glyceraldehyde, was confirmed to be a component

of a phytotoxic glycopeptide from potato plants infected with

Corynebacterium seDedonicum (Strobel, 1970), and the carboxyl

group of this acid was critical to the biological activity of

the toxin (Johnson and Strobel, 1970). Although it is a good

substrate for DS-Co, DL-glyceraldehyde has been demonstrated

to be a potent inhibitor for the carbon fixation of spinach

chloroplasts, and almost complete inhibition was observed at

10 mM concentration (Stokes and Walker, 1972). It was also

shown to inhibit phosphoribulose kinase or phosphoribose

isomerase (Bamberger and Avron, 1975), and the light

activation of ribulose bisphosphate carboxylase (Bahr and

Jensen, 1977) in intact chloroplasts. On the other hand,

glycolaldehyde was also reported to be more inhibitory than

DL-glyceraldehyde on carbon assimilation of spinach

chloroplasts (Sicher, 1984; Miller and Canvin, 1989).

Furthermore, glyoxylate inhibition of ribulose bisphosphate

carboxylase was also observed in intact, lysed, and

reconstituted chloroplasts (Campbell and Ogren, 1990). Under

the auspices of DS-Co, PEP may serve as a sink for

glyceraldehyde, glycolaldehyde, and glyoxylate for the

synthesis of beneficial molecules when toxic molecules are

inevitably produced in the plant cells, in addition to the

central role it plays in plant metabolism (Davies, 1979).







64

During microbial infection the cell may convert products of

glycolytic pathway to six carbon homologue of DAHP molecules

due to increased respiration after infection (Kahl, 1974;

Darvill and Albersheim, 1984).

2-Keto-3-deoxygalactonic acid has also been described

as a constituent of an extracellular polysaccharide of

Azotobacter vinelandii (Claus, 1965) and Vibrio

parahaemolvticus (Kondo et al., 1989), its higher plant

counterpart may await discovery as with the case of 2-keto-3-

deoxy-octonate (York et al., 1985). In fact, 3-deoxy-D-lyxo-2-

heptulosaric acid has also been identified as a cell wall

component in higher plants and a green alga (Stevenson et al.,

1988; Becker et al., 1989).

Multifunctionality

DS-Co may be a versatile enzyme that plays a different

role at different physiological condition. Ambiguity of

substrate utilization can be advantageous for the cell in

situations where it is appropriate for a family of unrelated

substrates to generate a family of related products. For

example, acetolactate synthase condenses two pyruvate

molecules or one pyruvate plus one ketobutyrate for the

biosynthesis of valine and isoleucine respectively. Fractional

product outputs of DS-Co may be dependent of developmental and

environmental impacts. Compartmentation may also dictate the

major catalytic reaction underway. For example, a form of DS-

Co could be compartmented in glyoxysomes, where glyoxylate







65

formation is a major specialized function via the glyoxylate

shunt. Thus, in castor bean seed tissue, glyxylate may be a

major source of a-ketoglutarate via DS-Co, whereas E4P and/or

G3P may be the source of cytoplasmic aromatic amino acids in

other tissues via DS-Co.










CHAPTER IV
KDOP SYNTHASE



Introduction

3-Deoxy-D-manno-octulosonate-8-phosphate (KDOP) synthase

is an enzyme of lipopolysaccharide biosynthesis (Schmidt and

Jann, 1983), thought until recently to be restricted to the

purple-bacteria group of prokaryotes. Preliminary data showing

the presence in potato tubers of KDOP synthase has been

reported (Morris et al, 1989). These results are consistent

with recent reports (York et al., 1985; Stevenson et al.,

1988) of the presence of 3-deoxy-D-manno-octulosonate and

related compounds in the cell walls of a variety of plants.

Owing to substrate ambiguity, this KDOP synthase possessed

weak activity as 3-deoxy-D-arabino-heptulosonate-7-phosphate

(DAHP) synthase, the initial catalytic step of aromatic amino

acid biosynthesis. The plastid-localized DS-Mn and cytosol-

localized DS-Co isozymes of DAHP synthase either did not

catalyze the KDOP synthase reaction at all (DS-Mn), or did so

very poorly (DS-Co). KDOP synthase from potato is demonstrated

more directly after its fractionation free of DS-Mn and DS-Co.

KDOP synthase from spinach was quite active and was selected

for partial purification and further characterization. The

general presence of KDOP synthase in crude extracts of a

variety of higher plants was readily demonstrated.






67

Materials and Methods

Plant Material

Spinach, Idaho potatoes, broccoli, carrots, cucumbers,

onions, sweet potatoes, apples, and cabbages were purchased

from a local supermarket. Suspension-cultured cells of

Nicotiana silvestris (EE cells) that had been maintained

continuously in exponential-phase growth (Bonner et al., 1988)

were used. All plant material was washed with deionized water,

frozen with liquid nitrogen, and ground to a fine powder by

use of a Waring blender. The powders were stored at -700C

prior to extract preparation.

Plant Extract Preparation and Enzyme Assays

All procedures were carried out at 0-40C. A 45-g amount

of powder of spinach or potato was mixed with 30 ml of buffer

A (50 mM K phosphate, pH 7.2, containing 0.5% P-

mercaptoethanol) and thawed at room temperature. The extract

was clarified by centrifugation at 29,000 x g for 30 min. and

filtered through Miracloth. A one-tenth volume of 2% protamine

sulfate in buffer A was slowly added to the extract and

stirred for 10 min.. The precipitate was removed by

centrifugation at 29,000 x g for 20 min.

Spinach and potato extracts used for column

chromatography were further treated as follows. The foregoing

supernatants were brought to 70% (spinach) or to 60% (potato)

of saturation with finely ground ammonium sulfate and stirred

for 10 min. The protein precipitate was collected by







68

centrifugation at 29,000 x g for 20 min. and resuspended in a

minimal volume of buffer B (10 mM EPPS, pH 7.5, and 50 mM

KCI). Desalting was accomplished by passage through Sephadex

G-25 (PD-10) columns equilibrated with buffer B according to

the manufacturer's instructions. The eluate was used for the

following chromatography.

DE-52 Column ChromatograDhv of Plant Extracts

A 150-mg (84-mg for potato) of spinach protein was loaded

onto a DE-52 cellulose column (1.5 x 19 cm) equlibribrated

with buffer B. The column was washed with 3 bed volumes of the

buffer before a 400 ml gradient (50-300 mM KC1) in buffer B

was applied. For the potato extracts a 400 ml gradient (50-500

mM KC1) was employed. The flow rate was 30 ml/hr, and

fractions of 2.9 ml were collected. The enzyme assays for KDOP

synthase, DS-Mn, and DS-Co were carried out as described

previously (Morris et al, 1989).

Thin-layer Chromatography

The enzymatic product of KDOP synthase was spotted onto

a cellulose-coated plastic plate (Chromagram of Eastman Kodak

Company, Rochester, New York). Ascending chromatography was

performed using an ethyl acetate/pyridine/acetic acid/water

(4:5:1:4 by volume) mixture as the solvent. The chromatogram

was dried and sprayed with reagent A (one volume of 0.1 M

sodium periodate in water mixed with 9 volume of acetone) and

after 15 min, with reagent B (One volume of ethylene glycol

and 1/10 volume of 10 M sulfuric acid mixed with 19 volumes of







69

acetone. After 30 min the plate was sprayed with reagent C (2%

of 2-thiobarbituric acid prepared in 96% hot ethanol) as

described by Brade and Galanos (1983).

Biochemicals

Sodium D-E4P, sodium D-ribose-5-P, sodium D-A5P, PEP

(monocyclohexylammonium salt), D-arabinose, L-arabinose, D-

ribose, 2-keto-3-deoxy-octonate (ammonium salt), protamine

sulfate (from salmon), and buffers (EPPS, PIPES, MES, HEPES,

BTP, CAPS, and Bicine) were obtained from Sigma (St. Louis,

MO). DTT was purchased from Research Organics, (Cleveland,

OH), and DE-52 anion exchanger was obtained from Whatman, Inc.

(Clifton, NJ). PD-10 columns were from Pharmacia (Piscataway,

NJ).



Results

KDOP Synthase in Potato

In previous work with potato, the elution profile of KDOP

synthase from DEAE cellulose overlapped with that of the

plastid-localized isozyme of DAHP synthase, DS-Mn. Under

conditions where DTT (required by DS-Mn) and divalent cation

(required by DS-Co) were omitted from reaction mixtures, a

single minor peak of apparent DAHP synthase activity was

discerned at an elution position between the DS-Mn and DS-Co

isozymes. This enzyme proved to be much more active when E4P

was replaced with A5P, and therefore appeared to be KDOP

synthase.







70

In this study the conditions used for application of the

salt gradient were modified to accomplish complete separation

of DS-Mn and KDOP synthase (Fig. 2-1). The leading and peak

fractions of KDOP synthase were also completely separated from

DS-Co. KDOP synthase eluted at 0.11 M KC1.

KDOP Svnthase in Spinach

Entirely comparable results were obtained following DEAE-

cellulose chromatography of spinach extract (Fig. 2-2). DS-Mn

eluted in the wash fractions, with KDOP synthase eluting

earlier in the salt gradient than DS-Co. In spinach the

relative amounts of DS-Mn and KDOP synthase exceeded those

found in potato, while the relative amount of DS-Co was less.

Given the better separation achieved and the greater activity

of KDOP synthase in spinach, further characterization was

carried out with the spinach enzyme.

Activity of KDOP synthase was followed as a function of

incubation temperature (Fig. 4-1) in 20 min assays. A rather

high temperature optimum of about 53C was obtained. PEP was

found to confer a striking degree of thermostability (Table 4-

1). In the absence of PEP, KDOP synthase was unstable at even

270C. This contrasts with the complete thermostability

attained in the presence of PEP in the vicinity of 50C. The

pH optimum for activity (Fig. 4-2) was determined at both 37C

and 500C. Activity increased sharply with pH up to about 6.2,

and declined progressively (but to a modest extent) as pH was

raised up to 10. Similar temperature and pH optima were found






71

for KDOP synthase from potato tuber and suspension-cultured

cells of Nicotiana silvestris (unpublished data). Divalent

cations were not required for KDOP synthase activity (in

contrast to DS-Co). KDOP synthase activity was also not

stimulated by divalent cations (in contrast to DS-Mn) in

experiments where 1 mM concentrations of Mg*, Mn", Co*, Ni*,

Fe*, Ca", and Ba were tested. Unlike the enzyme from

Pseudomonas aerucinosa (Levine and Racker, 1959), KDOP

synthase from spinach was not inhibited by 1 mM EDTA.

Partially purified spinach KDOP synthase was used to

construct substrate saturation curves for A5P and PEP. Simple

first-order kinetics were observed for ASP, and a double

reciprocal replot gave a Km value of 0.27 mM (Fig. 4-5). The

affinity for PEP was so high that it was difficult to obtain

accurate data points at very low PEP concentrations,

especially without a continuous assay. The Km value for PEP

was estimated to be about 35 AM. The substrate specificity of

KDOP synthase was examined. PEP could not be replaced by

pyruvate, thus ruling out the possibility that the enzyme

might be a reversible aldolase. KDOP synthase was able to

utilize E4P and R5P 24% and 7%, respectively, as well as D-

arabinose-5-P when these substrates were compared at 3 mM

concentrations. No activity was detected when D-arabinose, L-

arabinose, or D-ribose were used as substrates in combination

with PEP.







72

The product of KDOP synthase was identified as KDOP by

means of thin-layer chromatography (Fig. 4-3). When crude

extract was incubated with PEP and A5P, KDOP formed was

rapidly dephosphorylated to KDO. Thus, a mixture of KDOP and

KDO is visualized after 10 min of reaction in lane 2. The KDO

migrated to the same position as did authentic KDO (Rf= 0.42).

The Rf value for KDOP was 0.21. In samples taken after elapsed

reaction times of 30 minutes or greater, only KDO was

visualized. On the other hand, partially purified enzyme

recovered from anion-exchange chromatography (see Fig. 2-3)

exhibited substantial separation from KDOP phosphatase

activity. Thus, in lane 7, the majority of the reaction

product was KDOP when partially purified enzyme was used, even

after 7 hours of incubation of the reaction mixture.

Data shown in Fig. 4-4 indicate that on the basis of the

kinetics of the oxidation by periodate of KDOP formed by KDOP

synthase, the relative configuration of hydroxy groups at C-4

and C-5 of the molecule is cis. This orientation makes KDOP

more prone to rapid oxidation by periodate than is DAHP, a

molecule whose hydroxy groups at C-4 and C-5 exhibit a trans

configuration (Ghalambor et al., 1966). The configuration of

the eight-carbon product is hence inferred to be identical to

that of bacterial KDOP.

As expected if KDOP synthase does not function as a DAHP

synthase in vivo, aromatic amino acids, singly or in

combination, did not feedback inhibit KDOP synthase.






73

Chorismate, prephenate, or arogenate (intermediary

metabolites) having precedence as allosteric agents for DAHP

synthase, also failed to inhibit the activity of KDOP

synthase.

Other Plants

KDOP synthase can be assayed readily in crude extracts of

higher plants. Table 4-2 shows the results of assays carried

out with a variety of higher plants other than potato or

spinach. Except for apple and cabbage, KDOP synthase activity

was measured in all of the other plant species indicated. The

result obtained showing that KDOP synthase levels in potato

tissue assayed two days after mechanical wounding was not

elevated contrasts with the elevated levels induced for both

DS-Mn and DS-Co (Morris et al., 1989).











Table 4-1. Thermostability of spinach KDOP synthase


% of control activity

Thermal treatment + PEP PEP



270C 100 82

370C 105 72

470C 102 62

57C 92 37



Partially purified enzyme (Fig. 2-2) was incubated with
or without 1.1 mM PEP at the indicated temperature for 30 min
and removed to an ice bath prior to assay at 37C under
standard conditions.









Table 4-2. General presence of KDOP synthase in higher
plants


Higher plant"


Apple


Brocolli

Cabbage

Carrot

Cucumber

Onion

Sweet Potato


Potato, day 0c

day 2c

Spinach

Tobacco (EE cells)d


Specific activity


a Powders prepared as given in Methods were dissolved
(3g/2ml) in Buffer A.
b nmol KDOP/min/mg protein.
c Extracts were prepared from the mechanical wounding
experiment as described by Morris et al. (1989).
d Suspension-cultured cells of Nicotiana silvestris
continuously maintained in exponential-phase growth as
described by Bonner et al. (1988).


0.12


6.5

2.1

0.58

0.27

0.55

0.54

1.7

1.0







0 1 go
too 0 0

a1 00



x 4X
S IC) (


0 to n
ar) d'.
0.01 X#





14 0-4 -


4J 4)
x U)0
4) tv tri Ln ~

k r.





.$- a)
4J
041 -4 C:





(0 C) 4 k
4J 7 4-








to 4.1
043 r4 L

S.4a) 4J


V -74 00

*,74 .Qr-I0
0 4j

















rz. 0 E-4
$h4 ~ 4)
5 .40








Vr4~
4) 0
VOOq~
P F tf e
V $4
4)0
E-4 A -4
4) r-4
4) ,.1




a a



t; W m 9
-rq Ac r-I 41 0





4Ir~ ) to

4) 0 -r4 :3U
V 1-4 0




























































ol co 3
*: Sj 6


Ut

0
o




C-


4

LU


0 0


6tPsv









= c --4j
P4% to
4~1 %0%--J
0 >1Z*4

)) W
> 0o




'a;
0 4




E4


04
0 1 0 *






go 0 o%4
14 %0 44 EG~
LqL"










o 41 04

0~I~ I
4) U- 0

















a'q 4)- ~
*P4 W44 00
















0.) 0 .
Va r. 0.4
4 i0 r i















0d 0 0 4 v)
.0 ON A

0, 0 toE



0 4Q) to :% Y

r-I ,r

.r4 43 0 $4a~



V 00k ,
n 14


,Q Ul en

t~ O 43 O I
4) ed >I 1cq


0h
As ".4 go II~uIC
w :0to T4 0























0.


0

609 v
































KDOP



1 2 3 4 5 6 7






Fig. 4-3. Thin-layer chromatography of KDOP and KDO.
Lanes 1 to 6 were spotted with enzyme assay aliquots (5 1l)
obtained after 0, 10, 30, 60, 120, and 420 min of reaction
time, using desalted crude extracts from spinach. Lane 7 was
spotted with a 7-hr reaction mixture obtained by use of
partially purified KDOP synthase from spinach (Fig. 2-2).





























Fig. 4-4. Kinetics of oxidation of KDOP by periodate.
KDOP was formed as the reaction product of KDOP synthase, and
DAHP was formed as the reaction product of DS-Co in 1.6 ml
reaction mixtures. Authentic KDO was purchased from Sigma. The
enzyme reactions were stopped with 0.4 ml of 20% TCA and
protein was removed. Periodate oxidation was terminated with
excess arsenite at the times indicated.























100








50


PERIODATE OXIDATION TIME (min)















a

to






4J
'p










U)

0
a)

r.



0









4-)
0






r-'4

S.4

1-4
0
4)t










0 $4

00
'I

















'44
( r
U

















10.

p4>
.9.4 41
a)
u
r.(
01-














0
E
rz4
a)
Ul
0d
U

































(L.ulw IOWU) A


(uiw klowu)


A
T


,I








0






85

Discussion

Our initial finding of KDOP synthase in potato (Morris et

al., 1989) was unexpected because this enzyme has only been

described in gram-negative prokaryotes. However, with the

appreciation that KDO residues have been reported to be

present in the rhamno-galacturonan-II pectic polysaccharide of

a wide variety of plant cell walls (York et al., 1985;

Stevenson et al., 1988), the enzymatic formation of KDOP by

higher plants is not surprising. At this time a specific

functional role of KDOP in plant cell-wall architecture

analogous to its well-defined role in microbial

lipopolysaccharides is completely unknown.

KDOP synthase from spinach and potato was found to

exhibit substrate ambiguity, accepting E4P 24% as well as A5P

under conditions of substrate saturation. The cytosolic DAHP

synthase isozyme (DS-Co) also exhibits substrate ambiguity,

accepting A5P 8% as well as E4P under conditions of substrate

saturation. Such a degree of substrate ambiguity has not been

reported before for these enzymes from any source. In fact,

three isozymes of DAHP synthase were detected in pea by Rothe

et al. (1976), and in carrot by Suzich et al. (1985). One of

these activities might be KDOP synthase in view of the results

obtained in our investigation.











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