Planta (1984)160:78-83
P l a n t a 9 Springer-Verlag 1984
Purification and properties of 5-enolpyruvylshikimate 3-phosphate synthase from seedlings of Pisum sativum L. David M. Mousdale and John R. Coggins Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, UK
5-Enolpyruvylshikimate 3-phosphate synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19) from shoot tissue of pea seedlings was purified to apparent homogeneity by sequential ammonium-sulphate precipitation, ionexchange and hydrophobic-interaction chromatography and substrate elution from cellulose phosphate. Gel electrophoresis and gel-permeation chromatography showed that the purified enzyme was monomeric with molecular weight 50,000. The herbicide glyphosate was a potent inhibitor of the forward enzyme-catalyzed reaction. Abstract.
Key words: Amino acid, aromatic (biosynthesis) - 5-Enolpyruvylshikimate 3-phosphate synthase (3-phosphoshikimate 1-carboxyvinyltransferase)Glyphosate - Pisum (shikimate pathway) - Shikimate pathway.
observation that the enzyme from microbial sources is inhibited by micromolar concentrations of glyphosate (Amrhein et al. 1980; Steinrficken and Amrhein 1980; Boocock and Coggins 1983). Quoted unpublished results (Steinriicken and Amrhein 1980) suggest that EPSP synthase in extracts of higher-plant tissues is also sensitive to glyphosate inhibition. In Neurospora crassa (Burgoyne etal. 1969; Lumsden and Coggins 1977), certain other fungi (Ahmed and Giles 1969; Bode and Birnbaum 1981 a) and Euglena gracilis (Patel and Giles 1979) EPSP synthase occurs in a multifunctional enzyme with four other shikimate pathway enzyme activities: 3-dehydroquinate synthase (EC 4.6.1.3), 3-deC~OCH 2 H HO " z ' J y L o OH
c%
erythrose-4phosphate
.o.. c% NAD§
+
~= @OCH @0
The biosynthesis of aromatic amino acids in plants and micro-organisms proceeds by the shikimate pathway (Haslam 1974; see Fig. 1). Little detailed investigation of the enzymology of this pathway in higher plants has been accomplished and its invivo regulation is undefined (Gilchrist and Kosuge 1980). 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19) catalyzes the reversible addition of the enolpyruvyl moiety of phosphoenolpyruvate to shikimate 3-phosphate. Interest in EPSP synthase as a primary site of action of the broad-spectrum herbicide glyphosate (Nphosphonomethylglycine) has been focused by the Abbreviations: DEAE=diethylaminoethyl; EPSP=5-enolpyruvylshikimate 3-phosphate
OH
~
OH
Introduction
J OH
OH
)"
3- dehydr oquinaie
3- D e o x y - D - A r a b i n o -
CH2
0
Heptulosonic A c i d - 7 - P O 4
phosphoenolpyruvat e
CO 2
o'V'o. 6.
PEP
CO-
A "
.o-J~o. 6.
3-dehydrosbLkim~t e
CO2
sh~imat e
"o.
prephenate . ~ F
2
~
:| o" "~
c
shLkimat e 3-phosphate
CO2
FMN D
OH 5- eno]pyruv3tl- shikimat e-
B
~~
tyrosine
an t h r a n i [ at e -~tr)qo t ophan
(DH chorismate
3-phosphate
Fig. 1. Shikimate pathway for biosynthesis of aromatic amino acids in plants and micro-organisms. Principal enzymes mentioned in text: A, shikimate dehydrogenase; B, shikimate kinase; C, EPSP synthase; D, chorismate synthase
D.M. Mousdale and J.R. Coggins: 5-Enolpyruvylshikimate 3-phosphate synthase in Pisum sativum
hydroquinate dehydratase (EC 4.2.1.10), shikimate dehydrogenase (EC 1.1.1.25) and shikimate kinase (EC 2.7.1.71). In bacteria (Berlyn and Giles 1969), a blue-green alga, an alga and a moss (Berlyn et al. 1970) and Phaseolus mungo seedlings (Koshiba 1979) EPSP synthase is separable from other enzymes of the shikimate pathway. The monofunctional EPSP synthase from Escherichia coli has recently been purified to homogeneity in this laboratory (Lewendon and Coggins 1983). We have modified this procedure to purify the enzyme from pea seedlings and studied some of its molecular and kinetic properties. Material and methods Plant material. Seeds of Pisum sativum L. cv. Onward (Clause (UK) Ltd., Charvil, Berks., UK) were soaked in tap water for 16 h and planted in Fisons Levington multipurpose potting compost. Plants were grown at 23 ~ C with a 12-h photoperiod provided by Thorn-EMI (Hayes, Middlesex, UK) white fluorescent lamps (3.7 W m-Z). Enzyme extraction and purification. All steps were carried out at 4 ~ C. Step 1 : extraction and centrifugation. Shoots of 12-to 14-d-old seedlings were excised above the first leaf pair. Samples of 50 256 g fresh weight were homogenized for 30 s at full speed in a Waring blendor in 2 ml g x 50 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol Tris-HC1 (pH 7.5) containing 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM dithiothreitol (DTT), 1 mM benzamidine hydrochloride and 1 mM phenylmethanesulphonyl fluoride (buffer A). The brei was squeezed through two layers of cheesecloth and four layers of nylon cloth and the filtrate centrifuged at 80,000 g for 2 h. Step 2: ammonium-sulphate fraetionation. Powdered ammonium sulphate was slowly added to give a final concentration of 258 g1-1 (45% saturation). The solution was stirred for 30min and the precipitate removed by centrifugation at 12,000 g for 30 min. Ammonium sulphate was added to the supernatant to give a final concentration of 398 g1-1 (65% saturation) and the resulting precipitate collected by centrifugation at 12,000 g for 30 min. The precipitate was redissolved in buffer A and dialyzed against 2 x 600 ml buffer A for 16 h. Step 3: ion-exchange chromatography. The dialyzed fraction was loaded on to a column (12 cm long, 2.5 cm diameter) of diethylaminoethyl (DEAE)-Sephacel (Pharmacia, Hounslow, Middlesex, UK) equilibrated with 50 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol Tris-HC1 (pH 7.5) containing 1 mM EDTA, 0.1 m M DTT and 1 m M benzamidine hydrochloride (buffer B). The column was washed with buffer B until the absorbance at 280 nm was below 0.1 and then eluted with an increasing linear gradient (200 ml) of 0 to 100 mM KC1 in buffer B. Fractions with EPSP-synthase activity were combined and dialyzed against 2 x 600 ml buffer B for 16 h. Step 4: hydrophobic-interaction chromatography. The dialyzed fraction was brought to 106 g I-1 with solid ammonium sulphate and loaded on to a column (6 cm long, 1.5 cm diameter) of phenyl-Sepharose CL-4B (Pharmacia) equilibrated with 106g1-1 ammonium sulphate in buffer B. The column was washed with an equal volume of 106 g 1-1 ammonium sulphate
79
in buffer B and then eluted with a decreasing linear gradient (150 ml) of 106 to 0 g1-1 ammonium sulphate in buffer B. Fractions containing EPSP-synthase activity were combined and dialyzed against 2 x 6 0 0 m l 1 0 m M potassium citrate (pH 5.5) containing 0.1 mM DTT and 1 mM benzamidine hydrochloride (buffer C). Step5: cellulose-phosphate chromatography. The dialyzed fraction was loaded on to a column (4 cm long, 0.8 cm diameter) of cellulose phosphate P l l (Whatman Biochemicals, Maidstone, Kent, UK) equilibrated with buffer C. The column was washed with buffer C until the absorbance was below 0.005 and then eluted with 1 m M phosphoenolpyruvate in buffer C. When the absorbance at 280 nm was again below 0.005, the column was further eluted with 1 mM shikimate 3-phosphate and 1 mM phosphoenolpyruvate in buffer C. Fractions containing EPSP-synthase activity were combined and dialyzed against 4 x 600 ml buffer B. The enzyme preparation was redialyzed against 600 ml buffer B containing 50% (v/v) glycerol and stored at - 2 0 ~ C. Enzyme assays. The EPSP synthase was routinely assayed in the reverse direction by coupling the release of phosphoenolpyruvate to the pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) reactions (Boocock and Coggins 1983). Assay mixtures contained in a total volume of 1 ml: 100 mM potassium phosphate (pH 7.0), 2.5 mM ADP, 2.5 mM MgCI 2 0.1 mM N A D H , 50 gM EPSP (to initiate reaction) and 50 nkat pyruvate kinase and 42 nkat lactate dehydrogenase (Boehringer Chemical Corp., Lewes, East Sussex, UK). Oxidation of N A D H was monitored at 340 nm at 25 ~ C. The forward reaction was assayed in purified enzyme preparations by coupling the formation of EPSP to the chorismatesynthase (EC 4.6.1.4) reaction and monitoring chorismate formation at 275nm (e27s=2630 M -1 cm 1; Gibson 1970) at 25 ~ C (Boocock and Coggins 1983). Assay mixtures contained in a total volume of 1 ml : 50 mM triethanolamine hydrochloride-KOH (pH 7.0), 50 mM KC1, 2.5 mM MgC12, 10 gM NADPH, 10 gM flavine mononucleotide (FMN), 0.5 mM phosphoenolpyruvate (to initiate assay), 0.5 m M shikimate 3-phosphate and 0.08 nkat partially purified chorismate synthase from Neurospora crassa (the gift of Mr. M.R. Boocock, Department of Biochemistry, University of Glasgow). Shikimate dehydrogenase was assayed by following shikimate-dependent N A D P § reduction at 340 nm (Lumsden and Coggins 1977). Shikimate kinase was assayed by coupling the liberation of ADP to the pyruvate-kinase and lactate-dehydrogenase reactions (Smith and Coggins 1983). Chorismate synthase was assayed by following chorismate formation at 275 nm (Boocock and Coggins 1983). Shikimate 3-phosphate, isolated from Aerobacter aerogenes strain A.T.C.C. 25597, was the gift of Dr. G.A. Nimmo, Department of Biochemistry, University of Glasgowl The barium salt of EPSP, prepared enzymatically from shikimate-3-phosphate, was the gift of Ms. A. Lewendon, Department of Biochemistry, University of Glasgow; the potassium salt was prepared by adding a fivefotd excess of K2SO 4 and removing the precipitated BaSO 4 by centrifugation. Analytical-grade glyphosate was the gift of Dr. S.M. Ridley (I.C.I. plc. Jealott's Hill Research Station, Bracknell, Berkshire, UK). Protein determination. Protein was determined by Coomassiedye binding (Bradford 1976). With unpurified extracts, protein determinations were made on 10% (w/v) trichloroacetic-acid precipitates redissolved in 0.1 M NaOH. Polyacrylamide-gel electrophoresis. Electrophoresis was performed by the method of Davis (1964) in 7% polyacrylamide
80
D.M. Mousdale and J.R. Coggins : 5-Enolpyruvylshikimate 3-phosphate synthase in Pisum sativum
sulted in a purification of greater than 200-fold with a 60% recovery of the initial EPSP-synthase activity (Table 1). The final purification step used combined substrate elution of the enzyme from cellulose phosphate at pH 5.5 (Fig. 4). After this step, the purification achieved was greater than 3,000-fold with a recovery of approx. 20% (Table 1).
gels at 4 ~ C. Electrophoresis in the presence of sodium dodecyl sulphate was performed by the method of Laemmli (1970) with 3% stacking and 10% running gels. Protein staining was with Coomassie Blue G250. Gel-permeation chromatography. Gel-permeation chromatography was performed on a TSK G2000 SW column (60 cm long, 0.75 cm diameter) (LKB, South Croydon, UK) at room temperature with 0.067 M potassium phosphate (pH 6.8) as mobile phase. Injections (2 gg each of calibration proteins, 0.8 gg of purified EPSP synthase) were made in 25 gl. The flow rate of 0.5 ml rain-1 was provided by a model 303 pump (Gilson France S.A., Villiers-le-Bel, France). The column eluate was monitored at 215 nm with a LC-UV detector (Pye Unicam, Cambridge, UK) fitted with a 8 gl flow cell.
Homogeneity of purified EPSP synthase. The purified enzyme, after cellulose-phosphate chromatography, was electrophoretically homogeneous; when stained with Coomassie Blue, polyacrylamide gels showed a single band, Rf0.52. In the presence of sodium dodecyl sulphate, a single polypeptide was observed corresponding to a molecular weight of 50,000 (Fig. 5a). Gel-permeation chromatography on TSK G2000 SW indicated a molecular weight of 44,000 (Fig. 5b). Purified enzyme preparations were devoid of shikimate-dehydrogenase and shikimate-kinase activities and were inactive in assays for chorismate
Results
Purification of EPSP synthase. The 80,000-g supernatants from seedling tissue extracts contained EPSP synthase at a specific activity of 0.0250.05 nkat m g - ~ protein. Ammonium-sulphate fractionation gave a three- to four-fold purification (Table 1). Sequential chromatography on DEAESephacel (Fig. 2) and phenyl-Sepharose (Fig. 3) re-
Table 1. Purification of EPSP synthase from shoot tissue of pea seedlings Purification steps
Volume (ml)
Protein (rag m l - 1)
Activity (nkat m l - 1)
Activity (nkat)
Yield (%)
Specific activity Purification (nkat mg 1 protein) (-fold)
80,000-g centrifugation Ammonium-sulphate precipitation DEAE-Sephacel Phenyl-Sepharose Cellulose phosphate
380 118
3.4 2.9
0.097 0.286
37.0 33.7
100 91.2
0.03 0.10
0.76 0.125 0.006
0.551 0.810 0.567
29.2 24.3 8.5
78.9 65.7 23
0.72 6.5 94.5
53 30 15
1 3.3 24 217 3150
2.0
T'"
1,0"
50......,'"',.....,.-,~
2,0. 40-
v
-0.4
~
~~
5.0-
~
30
,~ 20 o
9"9 i ..~
2
~o~
,"""
i
i
I0
20
AI
J
5o
ao
Ar
J
50 00 Fraction
Ii
70
J
8o
10
9
~
90
ido
1100
3
0-
0 0
10
20
30
40
50
60
J 70
0
Fraction
Fig. 2. Chromatography of EPSP synthase on DEAE-Sephacel. Sample loading: 33.7 nkat EPSP synthase in extract after ammonium-sulphate fractionation. Flow rate: 1 ml min-1. Fractions: 5 ml. (A), EPSP-synthase activity; (o), absorbance at 280 nm; (e), conductivity. Linear gradient 0 to 100 mM KC1 begun at arrow Fig. 3. Chromatography of EPSP synthase on phenyl-Sepharose. Sample loading: 29.2 nkat EPSP synthase in extract after DEAESephacel step. Flow rate: 1 ml min-1. Fractions: 5 ml. (A), EPSP-synthase activity; (o), absorbance at 280 nm; (o), conductivity. Linear gradient 106 to 0 g 1 1 ammonium sulphate begun at arrow
D.M. Mousdale and J.R. Coggins: 5-Enolpyruvylshikimate 3-phosphate synthase in Pisum sativum
81
Table 2. Michaetis constants for EPSP-synthase substrates
0,2
0,2~
Substrate
Co-substrate (concentration)
Km
EPSP"
Phosphate (t00 raM) EPSP (50 ~M) Phosphoenolpyruvate (100 gM) Shikimate 3-phosphate (100 gM)
5.2_+2.8 p,M"
E
o oJ
Phosphate b
o
o
o
lo
20
;o
Fraction
Fig. 4. Chromatography of EPSP synthase on cellulose phosphate. Sample loading: 24.3 nkat EPSP synthase in extract after phenyl-Sepharose step. Flow rate: 15 mlh -t (loading and washing), 7 ml h-1 (elution). (A), EPSP-synthase activity; (o), absorbance at 280 nm. A t f i r s t arrow elution with 1 mM phosphoenolpyruvate begun; at second arrow elution with i mM phosphoenolpyruvate and 1 mM shikimate 3-phosphate
0,8
Shikimate 3-phosphate b Phosphoenolpyruvateb
3.7 + 1.9 mM 7.7_+0.8gM 5.2_+0.8 gM
a Buffer system: 100 mM phosphate (pH 7.0) b Buffer system : 50 mM triethanolamine hydrochloride-KOH (pH 7.0) c Mean + SD (three separate enzyme preparations)
500
~Io
(A)
g Ls
~0,6
=o
400
300
= 0.4
T
~3
200
>~
0,2
-~ I
0
[
~0
50
I
60
I
I
70
1oo
90
80
22
JO 0 " " ' o / O
o
2E 20
~
O"
o:1 [Phosphoeno[pyruvate] -1
o'.2 (#M-1 )
Fig. 6. Lineweaver-Burk plot of EPSP synthase with phosphoenolpyruvate as substrate. Co-substrate: 100gM shikimate 3-phosphate. (e), no glyphosate; (o), 5 p.M glyphosate o_
0
30
40
50
50
70
Molecular weight (x 10-3)
Fig. 5. A Polyacrylamide-gel electrophoresis of purified EPSP synthase in the presence of sodium dodecyl sulphate. Molecular-weight calibration proteins: 1, bovine erythrocyte carbonic anhydrase (29,000); 2, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (36,000); 3, rabbit muscle aldolase (40,000); 4, bovine liver glutamate dehydrogenase (53,000); 5, bovine liver catalase (60,000); 6, bovine serum albumin (68,000). B Gel-permeation chromatography. Molecular-weight calibration proteins: l, horse liver cytochromec (12,500); 2, sperm-whale myoglobin (17,000); 3, chicken ovalbumin (45,000); 4, bovine serum albumin (68,000)
synthase using N A D P H or N A D H with F M N or flavine-adenine dinucleotide ( F A D ) - the coenzyme requirements for higher-plant chorismate synthases have not been defined (Gilchrist and Kosuge 1980).
Kinetic parameters of EPSP synthase. The specific activity of the purified EPSP-synthase preparations was approx. 95 nkat rag-1 protein in the reverse direction and 192 nkat mg -1 protein in the forward direction. Between 85 and 90% of the activity was retained during 30 d storage at - 2 0 ~ C. Estimates of the Michaelis constants for EPSPsynthase substrates derived from kinetic analyses with the purified enzyme are given in Table 2. Glyphosate was a potent inhibitor of the forward enzyme-catalyzed reaction. It was competitive with respect to phosphoenolpyruvate and at 100 pM shikimate 3-phosphate its K~ was 80 nM (Fig. 6). Discussion The purification of EPSP synthase from pea seedlings as a monomeric enzyme of molecular weight 50,000 indicates that the enzyme from this source
82
D.M. Mousdale and J.R. Coggins: 5-Enolpyruvylshikimate 3-phosphate synthase in Pisum sativum
is very similar in gross structure to that purified from E. coli (Lewendon and Coggins 1983). For several other bacterial species, density-gradientcentrifugation analysis has yielded estimates of 33-45,000 for the molecular weight of unpurified EPSP synthase (Berlyn and Giles 1969). The molecular weight of the purified E. coli EPSP synthase was shown to be 55,000 by gel-permeation chromatography (Lewendon and Coggins 1983). By the same method, the unpurified enzyme from etiolated Phaseolus mungo seedlings was shown to have a molecular weight of 44,000 (Koshiba 1979) and the unpurified enzymes from several species of yeasts were found to have molecular weights in the range 39-44,000 (Bode and Birnbaum 1981 a). The sub-unit molecular weight of the E. coli enzyme was estimated by sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis to be 49,000 (Lewendon and Coggins 1983). The EPSP synthase from pea seedlings is thus similar to the enzyme from bacteria in that it can be purified as a monofunctional enzyme lacking other shikimate-pathway activities. In bacteria, the enzymes responsible for chorismate formation are separable (Berlyn and Giles 1969), the exception being Bacillus subtilis in which dehydroquinate synthase occurs as an aggregate with chorismate synthase, and shikimate kinase occurs in a trifunctional complex with 2-keto-3-deoxy-D-arabinoheptonate phosphate synthase (EC 4.1.2.15) and chorismate mutase (EC 5.4.99.5) (Huang et al. 1975; Hasan and Nester 1978). Shikimate dehydrogenase and dehydroquinate dehydratase from higher plants have been reported to copurify, although purification to homogeneity of the enzyme or enzyme aggregate has not been accomplished (Boudet and Lecussan 1974; Boudet et al. 1975; Koshiba 1978). Both enzyme activities are carried on a single polypeptide in the moss Physcomitrella patens (Polley 1978). Detailed investigation of shikimate-pathway enzymes may elucidate biochemical evolutionary relationships between micro-organisms, lower plants and higher plants. Estimates of Michaelis constants for pea seedling EPSP synthase (Table 2) are in broad agreement with those derived from detailed kinetic studies of the EPSP-synthase activity in the purified arom multienzyme complex from Neurospora crassa (Boocock and Coggins 1983). At pH 7, the K m values for inorganic phosphate exhibited by both enzymes appear to be substantially higher than the K,, values for the other three substrates. With the EPSP synthase purified 62-fold from the yeast Hansenula henricii, the K m values for shikimate 3-phosphate and phosphoenolpyruvate at
pH 5.5 were estimated to be 110 gM and 200 gM, respectively (Bode and Birnbaum 1981 b) which are substantially higher than the values for both the Neurospora and pea seedling enzymes. The effective inhibition of the forward EPSPsynthase-catalyzed reaction by micromolar concentrations of glyphosate confirms the indications from studies with microbial enzymes that this is a potential primary site of the herbicidal action (Amrhein et al. 1980; Steinrficken and Amrhein 1980; Boocock and Coggins 1983). It is particularly interesting that glyphosate, which is a competitive inhibitor with respect to phosphoenolpyruvate of both the Neurospora enzyme (Boocock and Coggins 1983) and the pea seedling enzyme, is a much more effective inhibitor of the latter. Thus the ratio Ki/Km for the pea seedling enzyme has the very low value of 0.015 while the corresponding ratio for the Neurospora enzyme is 0.31 (Boocock and Coggins 1983). Other shikimate-pathway enzymes are inhibited in vitro by glyphosate but this requires millimolar concentrations of the herbicide (Roisch and Lingens 1980; Rubin et al. 1982). This work was supported by grants from the Science and Engineering Research Council and I.C.I. plc. We would like to thank Dr. M.F. Hipkins (Department of Botany, University of Glasgow) for assistance with growing the plants.
References Ahmed, S.I., Giles, N.H. (1969) Organization of enzymes in the common aromatic synthetic pathway: evidence for aggregation in fungi. J. Bacteriol. 99, 231-237 Amrhein, N., Schab, J., Steinrficken, H.C. (1980) The mode of action of the herbicide glyphosate. Naturwissenschaften 67, 356-357 Berlyn, M.B., Ahmed, S.I., Giles, N.H. (1970) Organization of polyaromatic biosynthetic enzymes in a variety of photosynthetic organisms. J. Bacteriol. 104, 768-774 Berlyn, M.B., Giles, N.H. (1969) Organization of enzymes in the polyaromatic synthetic pathway: separability in bacteria. J. Bacteriol. 99, 222-230 Bode, R., Birnbaum, D. (1981 a) Aggregation und Trennbarkeit der Enzyme des Shikimat-Pathway bei Hefen. Z. Allg. Mikrobiol. 21,417-422 Bode, R., Birnbaum, D. (1981 b) Eigenschaften und Regulation der Enzyme des Shikimat-Pathway von Hansenula henricii. Biochem. Physiol. Pflanz. 176, 329-341 Boocock, M.R., Coggins, J.R. (1983) Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate. FEBS Lett. 154, 12%133 Boudet, A.M., Lecussan, R. (1974) G6n6ralit~ de l'association (5-d6shydroquinate hydro-lyase, shikimate: NADP + oxydo-r6ductase) chez les v~g6taux sup6rieurs. Planta 119, 71-79 Boudet, A.M., Lecussan, R., Boudet, A. (1975) Characterization and properties of 3-dehydroquinate hydrolyases in higher plants. Planta 124, 67-76 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing
D.M. Mousdale and J.R. Coggins: 5-Enolpyruvylshikimate 3-phosphate synthase in Pisum sativum the principle of protein-dye binding. Anal. Biochem. 72, 248-254 Burgoyne, L., Case, M.E., Giles, N.H. (1969) Purification and properties of the aromatic (arom) synthetic enzyme aggregate of Neurospora crassa. Biochim. Biophys. Acta 191, 452-462 Davis, B.J. (1964) Disc electrophoresis. II. Methods and applications to human serum proteins. Ann. N. Y. Acad. Sci. 121,404-427 Gibson, F. (1970) Preparation of chorismic acid. Methods Enzymol. 17, 362-364 Gilchrist, D.G., Kosuge, T. (1980) Aromatic amino acid biosynthesis and its regulation. In : The biochemistry of plants, vol. 5: Amino acids and derivatives, pp. 507-531, Miflin, B.J., ed. Academic Press, New York London Hasan, N., Nester, E.W. (1978) Dehydroquinate synthase in Bacillus subtilis: an enzyme associated with chorismate synthase and flavine reductase. J. Biol. Chem. 253, 4999-5004 Haslam, E. (1974) The shikimate pathway. Butterworths, London Huang, L., Montoya, A.L., Nester, E.W. (1975) Purification and characterization of shikimate kinase enzyme activity in Bacillus subtilis. J. Biol. Chem. 250, 7675-7661 Koshiba, T. (1978) Purification of two forms of the associated 3-dehydroquinate hydro-lyase and shikimate: NADP + oxidoreductase in Phaseolus mungo seedlings. Biochim. Biophys. Acta 522, 10-18 Koshiba, T. (1979) Organization of enzymes in the shikimate pathway of Phaseolus mungo seedlings. Plant Cell Physiol. 20, 667-670 Laemmli, U.K. (1970) Cleavage of structural proteins during
83
the assembly of the head of bacteriophage T4. Nature (London) 224, 680-685 Lewendon, A., Coggins, J.R. (1983) Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli. Biochem. J. 213, 187-191 Lumsden, J., Coggins, J.R. (1977) The subunit structure of the arom multienzyme complex of Neurospora crassa. Biochem. J. 161,599-607 Patel, V.B., Giles, N.H. (1979) Purification of the arom multienzyme aggregate from Euglena gracilis. Biochim. Biophys. Acta 567, 24-34 Polley, L.D. (1978) Purification and characterisation of 3-dehydroquinate hydrolase and shikimate oxidoreductase. Biochim. Biophys. Acta 526, 259-266 Roisch, U., Lingens, F. (1980) Einflug yon N-(Phosphonomethyl)-glycin auf das Wachstum nnd auf die Enzyme der Aromatenbiosynthese von Escherichia coli. Hoppe-Seyler's Z. Physiol. Chem. 361, 1049-1058 Rubin, J.L., Gaines, C.G., Jensen, R.A. (1982) Enzymological basis for herbicidal action of glyphosate. Plant Physiol. 70, 833 839 Smith, D.D.S., Coggins, J.R. (1983) Isolation of a bifunctional domain from the pentafunctional arom enzyme complex of Neurospora crassa. Biochem. J. 213, 405-415 Steinrficken, H.C., Amrhein, N. (1980) The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic acid-3phosphate synthase. Biochem. Biophys. Res. Commun. 94, 1207-1212 Received 29 July; accepted 12 September 1983
P l a n t a 9 Springer-Verlag 1984
Purification and properties of 5-enolpyruvylshikimate 3-phosphate synthase from seedlings of Pisum sativum L. David M. Mousdale and John R. Coggins Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, UK
5-Enolpyruvylshikimate 3-phosphate synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19) from shoot tissue of pea seedlings was purified to apparent homogeneity by sequential ammonium-sulphate precipitation, ionexchange and hydrophobic-interaction chromatography and substrate elution from cellulose phosphate. Gel electrophoresis and gel-permeation chromatography showed that the purified enzyme was monomeric with molecular weight 50,000. The herbicide glyphosate was a potent inhibitor of the forward enzyme-catalyzed reaction. Abstract.
Key words: Amino acid, aromatic (biosynthesis) - 5-Enolpyruvylshikimate 3-phosphate synthase (3-phosphoshikimate 1-carboxyvinyltransferase)Glyphosate - Pisum (shikimate pathway) - Shikimate pathway.
observation that the enzyme from microbial sources is inhibited by micromolar concentrations of glyphosate (Amrhein et al. 1980; Steinrficken and Amrhein 1980; Boocock and Coggins 1983). Quoted unpublished results (Steinriicken and Amrhein 1980) suggest that EPSP synthase in extracts of higher-plant tissues is also sensitive to glyphosate inhibition. In Neurospora crassa (Burgoyne etal. 1969; Lumsden and Coggins 1977), certain other fungi (Ahmed and Giles 1969; Bode and Birnbaum 1981 a) and Euglena gracilis (Patel and Giles 1979) EPSP synthase occurs in a multifunctional enzyme with four other shikimate pathway enzyme activities: 3-dehydroquinate synthase (EC 4.6.1.3), 3-deC~OCH 2 H HO " z ' J y L o OH
c%
erythrose-4phosphate
.o.. c% NAD§
+
~= @OCH @0
The biosynthesis of aromatic amino acids in plants and micro-organisms proceeds by the shikimate pathway (Haslam 1974; see Fig. 1). Little detailed investigation of the enzymology of this pathway in higher plants has been accomplished and its invivo regulation is undefined (Gilchrist and Kosuge 1980). 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19) catalyzes the reversible addition of the enolpyruvyl moiety of phosphoenolpyruvate to shikimate 3-phosphate. Interest in EPSP synthase as a primary site of action of the broad-spectrum herbicide glyphosate (Nphosphonomethylglycine) has been focused by the Abbreviations: DEAE=diethylaminoethyl; EPSP=5-enolpyruvylshikimate 3-phosphate
OH
~
OH
Introduction
J OH
OH
)"
3- dehydr oquinaie
3- D e o x y - D - A r a b i n o -
CH2
0
Heptulosonic A c i d - 7 - P O 4
phosphoenolpyruvat e
CO 2
o'V'o. 6.
PEP
CO-
A "
.o-J~o. 6.
3-dehydrosbLkim~t e
CO2
sh~imat e
"o.
prephenate . ~ F
2
~
:| o" "~
c
shLkimat e 3-phosphate
CO2
FMN D
OH 5- eno]pyruv3tl- shikimat e-
B
~~
tyrosine
an t h r a n i [ at e -~tr)qo t ophan
(DH chorismate
3-phosphate
Fig. 1. Shikimate pathway for biosynthesis of aromatic amino acids in plants and micro-organisms. Principal enzymes mentioned in text: A, shikimate dehydrogenase; B, shikimate kinase; C, EPSP synthase; D, chorismate synthase
D.M. Mousdale and J.R. Coggins: 5-Enolpyruvylshikimate 3-phosphate synthase in Pisum sativum
hydroquinate dehydratase (EC 4.2.1.10), shikimate dehydrogenase (EC 1.1.1.25) and shikimate kinase (EC 2.7.1.71). In bacteria (Berlyn and Giles 1969), a blue-green alga, an alga and a moss (Berlyn et al. 1970) and Phaseolus mungo seedlings (Koshiba 1979) EPSP synthase is separable from other enzymes of the shikimate pathway. The monofunctional EPSP synthase from Escherichia coli has recently been purified to homogeneity in this laboratory (Lewendon and Coggins 1983). We have modified this procedure to purify the enzyme from pea seedlings and studied some of its molecular and kinetic properties. Material and methods Plant material. Seeds of Pisum sativum L. cv. Onward (Clause (UK) Ltd., Charvil, Berks., UK) were soaked in tap water for 16 h and planted in Fisons Levington multipurpose potting compost. Plants were grown at 23 ~ C with a 12-h photoperiod provided by Thorn-EMI (Hayes, Middlesex, UK) white fluorescent lamps (3.7 W m-Z). Enzyme extraction and purification. All steps were carried out at 4 ~ C. Step 1 : extraction and centrifugation. Shoots of 12-to 14-d-old seedlings were excised above the first leaf pair. Samples of 50 256 g fresh weight were homogenized for 30 s at full speed in a Waring blendor in 2 ml g x 50 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol Tris-HC1 (pH 7.5) containing 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM dithiothreitol (DTT), 1 mM benzamidine hydrochloride and 1 mM phenylmethanesulphonyl fluoride (buffer A). The brei was squeezed through two layers of cheesecloth and four layers of nylon cloth and the filtrate centrifuged at 80,000 g for 2 h. Step 2: ammonium-sulphate fraetionation. Powdered ammonium sulphate was slowly added to give a final concentration of 258 g1-1 (45% saturation). The solution was stirred for 30min and the precipitate removed by centrifugation at 12,000 g for 30 min. Ammonium sulphate was added to the supernatant to give a final concentration of 398 g1-1 (65% saturation) and the resulting precipitate collected by centrifugation at 12,000 g for 30 min. The precipitate was redissolved in buffer A and dialyzed against 2 x 600 ml buffer A for 16 h. Step 3: ion-exchange chromatography. The dialyzed fraction was loaded on to a column (12 cm long, 2.5 cm diameter) of diethylaminoethyl (DEAE)-Sephacel (Pharmacia, Hounslow, Middlesex, UK) equilibrated with 50 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol Tris-HC1 (pH 7.5) containing 1 mM EDTA, 0.1 m M DTT and 1 m M benzamidine hydrochloride (buffer B). The column was washed with buffer B until the absorbance at 280 nm was below 0.1 and then eluted with an increasing linear gradient (200 ml) of 0 to 100 mM KC1 in buffer B. Fractions with EPSP-synthase activity were combined and dialyzed against 2 x 600 ml buffer B for 16 h. Step 4: hydrophobic-interaction chromatography. The dialyzed fraction was brought to 106 g I-1 with solid ammonium sulphate and loaded on to a column (6 cm long, 1.5 cm diameter) of phenyl-Sepharose CL-4B (Pharmacia) equilibrated with 106g1-1 ammonium sulphate in buffer B. The column was washed with an equal volume of 106 g 1-1 ammonium sulphate
79
in buffer B and then eluted with a decreasing linear gradient (150 ml) of 106 to 0 g1-1 ammonium sulphate in buffer B. Fractions containing EPSP-synthase activity were combined and dialyzed against 2 x 6 0 0 m l 1 0 m M potassium citrate (pH 5.5) containing 0.1 mM DTT and 1 mM benzamidine hydrochloride (buffer C). Step5: cellulose-phosphate chromatography. The dialyzed fraction was loaded on to a column (4 cm long, 0.8 cm diameter) of cellulose phosphate P l l (Whatman Biochemicals, Maidstone, Kent, UK) equilibrated with buffer C. The column was washed with buffer C until the absorbance was below 0.005 and then eluted with 1 m M phosphoenolpyruvate in buffer C. When the absorbance at 280 nm was again below 0.005, the column was further eluted with 1 mM shikimate 3-phosphate and 1 mM phosphoenolpyruvate in buffer C. Fractions containing EPSP-synthase activity were combined and dialyzed against 4 x 600 ml buffer B. The enzyme preparation was redialyzed against 600 ml buffer B containing 50% (v/v) glycerol and stored at - 2 0 ~ C. Enzyme assays. The EPSP synthase was routinely assayed in the reverse direction by coupling the release of phosphoenolpyruvate to the pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) reactions (Boocock and Coggins 1983). Assay mixtures contained in a total volume of 1 ml: 100 mM potassium phosphate (pH 7.0), 2.5 mM ADP, 2.5 mM MgCI 2 0.1 mM N A D H , 50 gM EPSP (to initiate reaction) and 50 nkat pyruvate kinase and 42 nkat lactate dehydrogenase (Boehringer Chemical Corp., Lewes, East Sussex, UK). Oxidation of N A D H was monitored at 340 nm at 25 ~ C. The forward reaction was assayed in purified enzyme preparations by coupling the formation of EPSP to the chorismatesynthase (EC 4.6.1.4) reaction and monitoring chorismate formation at 275nm (e27s=2630 M -1 cm 1; Gibson 1970) at 25 ~ C (Boocock and Coggins 1983). Assay mixtures contained in a total volume of 1 ml : 50 mM triethanolamine hydrochloride-KOH (pH 7.0), 50 mM KC1, 2.5 mM MgC12, 10 gM NADPH, 10 gM flavine mononucleotide (FMN), 0.5 mM phosphoenolpyruvate (to initiate assay), 0.5 m M shikimate 3-phosphate and 0.08 nkat partially purified chorismate synthase from Neurospora crassa (the gift of Mr. M.R. Boocock, Department of Biochemistry, University of Glasgow). Shikimate dehydrogenase was assayed by following shikimate-dependent N A D P § reduction at 340 nm (Lumsden and Coggins 1977). Shikimate kinase was assayed by coupling the liberation of ADP to the pyruvate-kinase and lactate-dehydrogenase reactions (Smith and Coggins 1983). Chorismate synthase was assayed by following chorismate formation at 275 nm (Boocock and Coggins 1983). Shikimate 3-phosphate, isolated from Aerobacter aerogenes strain A.T.C.C. 25597, was the gift of Dr. G.A. Nimmo, Department of Biochemistry, University of Glasgowl The barium salt of EPSP, prepared enzymatically from shikimate-3-phosphate, was the gift of Ms. A. Lewendon, Department of Biochemistry, University of Glasgow; the potassium salt was prepared by adding a fivefotd excess of K2SO 4 and removing the precipitated BaSO 4 by centrifugation. Analytical-grade glyphosate was the gift of Dr. S.M. Ridley (I.C.I. plc. Jealott's Hill Research Station, Bracknell, Berkshire, UK). Protein determination. Protein was determined by Coomassiedye binding (Bradford 1976). With unpurified extracts, protein determinations were made on 10% (w/v) trichloroacetic-acid precipitates redissolved in 0.1 M NaOH. Polyacrylamide-gel electrophoresis. Electrophoresis was performed by the method of Davis (1964) in 7% polyacrylamide
80
D.M. Mousdale and J.R. Coggins : 5-Enolpyruvylshikimate 3-phosphate synthase in Pisum sativum
sulted in a purification of greater than 200-fold with a 60% recovery of the initial EPSP-synthase activity (Table 1). The final purification step used combined substrate elution of the enzyme from cellulose phosphate at pH 5.5 (Fig. 4). After this step, the purification achieved was greater than 3,000-fold with a recovery of approx. 20% (Table 1).
gels at 4 ~ C. Electrophoresis in the presence of sodium dodecyl sulphate was performed by the method of Laemmli (1970) with 3% stacking and 10% running gels. Protein staining was with Coomassie Blue G250. Gel-permeation chromatography. Gel-permeation chromatography was performed on a TSK G2000 SW column (60 cm long, 0.75 cm diameter) (LKB, South Croydon, UK) at room temperature with 0.067 M potassium phosphate (pH 6.8) as mobile phase. Injections (2 gg each of calibration proteins, 0.8 gg of purified EPSP synthase) were made in 25 gl. The flow rate of 0.5 ml rain-1 was provided by a model 303 pump (Gilson France S.A., Villiers-le-Bel, France). The column eluate was monitored at 215 nm with a LC-UV detector (Pye Unicam, Cambridge, UK) fitted with a 8 gl flow cell.
Homogeneity of purified EPSP synthase. The purified enzyme, after cellulose-phosphate chromatography, was electrophoretically homogeneous; when stained with Coomassie Blue, polyacrylamide gels showed a single band, Rf0.52. In the presence of sodium dodecyl sulphate, a single polypeptide was observed corresponding to a molecular weight of 50,000 (Fig. 5a). Gel-permeation chromatography on TSK G2000 SW indicated a molecular weight of 44,000 (Fig. 5b). Purified enzyme preparations were devoid of shikimate-dehydrogenase and shikimate-kinase activities and were inactive in assays for chorismate
Results
Purification of EPSP synthase. The 80,000-g supernatants from seedling tissue extracts contained EPSP synthase at a specific activity of 0.0250.05 nkat m g - ~ protein. Ammonium-sulphate fractionation gave a three- to four-fold purification (Table 1). Sequential chromatography on DEAESephacel (Fig. 2) and phenyl-Sepharose (Fig. 3) re-
Table 1. Purification of EPSP synthase from shoot tissue of pea seedlings Purification steps
Volume (ml)
Protein (rag m l - 1)
Activity (nkat m l - 1)
Activity (nkat)
Yield (%)
Specific activity Purification (nkat mg 1 protein) (-fold)
80,000-g centrifugation Ammonium-sulphate precipitation DEAE-Sephacel Phenyl-Sepharose Cellulose phosphate
380 118
3.4 2.9
0.097 0.286
37.0 33.7
100 91.2
0.03 0.10
0.76 0.125 0.006
0.551 0.810 0.567
29.2 24.3 8.5
78.9 65.7 23
0.72 6.5 94.5
53 30 15
1 3.3 24 217 3150
2.0
T'"
1,0"
50......,'"',.....,.-,~
2,0. 40-
v
-0.4
~
~~
5.0-
~
30
,~ 20 o
9"9 i ..~
2
~o~
,"""
i
i
I0
20
AI
J
5o
ao
Ar
J
50 00 Fraction
Ii
70
J
8o
10
9
~
90
ido
1100
3
0-
0 0
10
20
30
40
50
60
J 70
0
Fraction
Fig. 2. Chromatography of EPSP synthase on DEAE-Sephacel. Sample loading: 33.7 nkat EPSP synthase in extract after ammonium-sulphate fractionation. Flow rate: 1 ml min-1. Fractions: 5 ml. (A), EPSP-synthase activity; (o), absorbance at 280 nm; (e), conductivity. Linear gradient 0 to 100 mM KC1 begun at arrow Fig. 3. Chromatography of EPSP synthase on phenyl-Sepharose. Sample loading: 29.2 nkat EPSP synthase in extract after DEAESephacel step. Flow rate: 1 ml min-1. Fractions: 5 ml. (A), EPSP-synthase activity; (o), absorbance at 280 nm; (o), conductivity. Linear gradient 106 to 0 g 1 1 ammonium sulphate begun at arrow
D.M. Mousdale and J.R. Coggins: 5-Enolpyruvylshikimate 3-phosphate synthase in Pisum sativum
81
Table 2. Michaetis constants for EPSP-synthase substrates
0,2
0,2~
Substrate
Co-substrate (concentration)
Km
EPSP"
Phosphate (t00 raM) EPSP (50 ~M) Phosphoenolpyruvate (100 gM) Shikimate 3-phosphate (100 gM)
5.2_+2.8 p,M"
E
o oJ
Phosphate b
o
o
o
lo
20
;o
Fraction
Fig. 4. Chromatography of EPSP synthase on cellulose phosphate. Sample loading: 24.3 nkat EPSP synthase in extract after phenyl-Sepharose step. Flow rate: 15 mlh -t (loading and washing), 7 ml h-1 (elution). (A), EPSP-synthase activity; (o), absorbance at 280 nm. A t f i r s t arrow elution with 1 mM phosphoenolpyruvate begun; at second arrow elution with i mM phosphoenolpyruvate and 1 mM shikimate 3-phosphate
0,8
Shikimate 3-phosphate b Phosphoenolpyruvateb
3.7 + 1.9 mM 7.7_+0.8gM 5.2_+0.8 gM
a Buffer system: 100 mM phosphate (pH 7.0) b Buffer system : 50 mM triethanolamine hydrochloride-KOH (pH 7.0) c Mean + SD (three separate enzyme preparations)
500
~Io
(A)
g Ls
~0,6
=o
400
300
= 0.4
T
~3
200
>~
0,2
-~ I
0
[
~0
50
I
60
I
I
70
1oo
90
80
22
JO 0 " " ' o / O
o
2E 20
~
O"
o:1 [Phosphoeno[pyruvate] -1
o'.2 (#M-1 )
Fig. 6. Lineweaver-Burk plot of EPSP synthase with phosphoenolpyruvate as substrate. Co-substrate: 100gM shikimate 3-phosphate. (e), no glyphosate; (o), 5 p.M glyphosate o_
0
30
40
50
50
70
Molecular weight (x 10-3)
Fig. 5. A Polyacrylamide-gel electrophoresis of purified EPSP synthase in the presence of sodium dodecyl sulphate. Molecular-weight calibration proteins: 1, bovine erythrocyte carbonic anhydrase (29,000); 2, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (36,000); 3, rabbit muscle aldolase (40,000); 4, bovine liver glutamate dehydrogenase (53,000); 5, bovine liver catalase (60,000); 6, bovine serum albumin (68,000). B Gel-permeation chromatography. Molecular-weight calibration proteins: l, horse liver cytochromec (12,500); 2, sperm-whale myoglobin (17,000); 3, chicken ovalbumin (45,000); 4, bovine serum albumin (68,000)
synthase using N A D P H or N A D H with F M N or flavine-adenine dinucleotide ( F A D ) - the coenzyme requirements for higher-plant chorismate synthases have not been defined (Gilchrist and Kosuge 1980).
Kinetic parameters of EPSP synthase. The specific activity of the purified EPSP-synthase preparations was approx. 95 nkat rag-1 protein in the reverse direction and 192 nkat mg -1 protein in the forward direction. Between 85 and 90% of the activity was retained during 30 d storage at - 2 0 ~ C. Estimates of the Michaelis constants for EPSPsynthase substrates derived from kinetic analyses with the purified enzyme are given in Table 2. Glyphosate was a potent inhibitor of the forward enzyme-catalyzed reaction. It was competitive with respect to phosphoenolpyruvate and at 100 pM shikimate 3-phosphate its K~ was 80 nM (Fig. 6). Discussion The purification of EPSP synthase from pea seedlings as a monomeric enzyme of molecular weight 50,000 indicates that the enzyme from this source
82
D.M. Mousdale and J.R. Coggins: 5-Enolpyruvylshikimate 3-phosphate synthase in Pisum sativum
is very similar in gross structure to that purified from E. coli (Lewendon and Coggins 1983). For several other bacterial species, density-gradientcentrifugation analysis has yielded estimates of 33-45,000 for the molecular weight of unpurified EPSP synthase (Berlyn and Giles 1969). The molecular weight of the purified E. coli EPSP synthase was shown to be 55,000 by gel-permeation chromatography (Lewendon and Coggins 1983). By the same method, the unpurified enzyme from etiolated Phaseolus mungo seedlings was shown to have a molecular weight of 44,000 (Koshiba 1979) and the unpurified enzymes from several species of yeasts were found to have molecular weights in the range 39-44,000 (Bode and Birnbaum 1981 a). The sub-unit molecular weight of the E. coli enzyme was estimated by sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis to be 49,000 (Lewendon and Coggins 1983). The EPSP synthase from pea seedlings is thus similar to the enzyme from bacteria in that it can be purified as a monofunctional enzyme lacking other shikimate-pathway activities. In bacteria, the enzymes responsible for chorismate formation are separable (Berlyn and Giles 1969), the exception being Bacillus subtilis in which dehydroquinate synthase occurs as an aggregate with chorismate synthase, and shikimate kinase occurs in a trifunctional complex with 2-keto-3-deoxy-D-arabinoheptonate phosphate synthase (EC 4.1.2.15) and chorismate mutase (EC 5.4.99.5) (Huang et al. 1975; Hasan and Nester 1978). Shikimate dehydrogenase and dehydroquinate dehydratase from higher plants have been reported to copurify, although purification to homogeneity of the enzyme or enzyme aggregate has not been accomplished (Boudet and Lecussan 1974; Boudet et al. 1975; Koshiba 1978). Both enzyme activities are carried on a single polypeptide in the moss Physcomitrella patens (Polley 1978). Detailed investigation of shikimate-pathway enzymes may elucidate biochemical evolutionary relationships between micro-organisms, lower plants and higher plants. Estimates of Michaelis constants for pea seedling EPSP synthase (Table 2) are in broad agreement with those derived from detailed kinetic studies of the EPSP-synthase activity in the purified arom multienzyme complex from Neurospora crassa (Boocock and Coggins 1983). At pH 7, the K m values for inorganic phosphate exhibited by both enzymes appear to be substantially higher than the K,, values for the other three substrates. With the EPSP synthase purified 62-fold from the yeast Hansenula henricii, the K m values for shikimate 3-phosphate and phosphoenolpyruvate at
pH 5.5 were estimated to be 110 gM and 200 gM, respectively (Bode and Birnbaum 1981 b) which are substantially higher than the values for both the Neurospora and pea seedling enzymes. The effective inhibition of the forward EPSPsynthase-catalyzed reaction by micromolar concentrations of glyphosate confirms the indications from studies with microbial enzymes that this is a potential primary site of the herbicidal action (Amrhein et al. 1980; Steinrficken and Amrhein 1980; Boocock and Coggins 1983). It is particularly interesting that glyphosate, which is a competitive inhibitor with respect to phosphoenolpyruvate of both the Neurospora enzyme (Boocock and Coggins 1983) and the pea seedling enzyme, is a much more effective inhibitor of the latter. Thus the ratio Ki/Km for the pea seedling enzyme has the very low value of 0.015 while the corresponding ratio for the Neurospora enzyme is 0.31 (Boocock and Coggins 1983). Other shikimate-pathway enzymes are inhibited in vitro by glyphosate but this requires millimolar concentrations of the herbicide (Roisch and Lingens 1980; Rubin et al. 1982). This work was supported by grants from the Science and Engineering Research Council and I.C.I. plc. We would like to thank Dr. M.F. Hipkins (Department of Botany, University of Glasgow) for assistance with growing the plants.
References Ahmed, S.I., Giles, N.H. (1969) Organization of enzymes in the common aromatic synthetic pathway: evidence for aggregation in fungi. J. Bacteriol. 99, 231-237 Amrhein, N., Schab, J., Steinrficken, H.C. (1980) The mode of action of the herbicide glyphosate. Naturwissenschaften 67, 356-357 Berlyn, M.B., Ahmed, S.I., Giles, N.H. (1970) Organization of polyaromatic biosynthetic enzymes in a variety of photosynthetic organisms. J. Bacteriol. 104, 768-774 Berlyn, M.B., Giles, N.H. (1969) Organization of enzymes in the polyaromatic synthetic pathway: separability in bacteria. J. Bacteriol. 99, 222-230 Bode, R., Birnbaum, D. (1981 a) Aggregation und Trennbarkeit der Enzyme des Shikimat-Pathway bei Hefen. Z. Allg. Mikrobiol. 21,417-422 Bode, R., Birnbaum, D. (1981 b) Eigenschaften und Regulation der Enzyme des Shikimat-Pathway von Hansenula henricii. Biochem. Physiol. Pflanz. 176, 329-341 Boocock, M.R., Coggins, J.R. (1983) Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate. FEBS Lett. 154, 12%133 Boudet, A.M., Lecussan, R. (1974) G6n6ralit~ de l'association (5-d6shydroquinate hydro-lyase, shikimate: NADP + oxydo-r6ductase) chez les v~g6taux sup6rieurs. Planta 119, 71-79 Boudet, A.M., Lecussan, R., Boudet, A. (1975) Characterization and properties of 3-dehydroquinate hydrolyases in higher plants. Planta 124, 67-76 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing
D.M. Mousdale and J.R. Coggins: 5-Enolpyruvylshikimate 3-phosphate synthase in Pisum sativum the principle of protein-dye binding. Anal. Biochem. 72, 248-254 Burgoyne, L., Case, M.E., Giles, N.H. (1969) Purification and properties of the aromatic (arom) synthetic enzyme aggregate of Neurospora crassa. Biochim. Biophys. Acta 191, 452-462 Davis, B.J. (1964) Disc electrophoresis. II. Methods and applications to human serum proteins. Ann. N. Y. Acad. Sci. 121,404-427 Gibson, F. (1970) Preparation of chorismic acid. Methods Enzymol. 17, 362-364 Gilchrist, D.G., Kosuge, T. (1980) Aromatic amino acid biosynthesis and its regulation. In : The biochemistry of plants, vol. 5: Amino acids and derivatives, pp. 507-531, Miflin, B.J., ed. Academic Press, New York London Hasan, N., Nester, E.W. (1978) Dehydroquinate synthase in Bacillus subtilis: an enzyme associated with chorismate synthase and flavine reductase. J. Biol. Chem. 253, 4999-5004 Haslam, E. (1974) The shikimate pathway. Butterworths, London Huang, L., Montoya, A.L., Nester, E.W. (1975) Purification and characterization of shikimate kinase enzyme activity in Bacillus subtilis. J. Biol. Chem. 250, 7675-7661 Koshiba, T. (1978) Purification of two forms of the associated 3-dehydroquinate hydro-lyase and shikimate: NADP + oxidoreductase in Phaseolus mungo seedlings. Biochim. Biophys. Acta 522, 10-18 Koshiba, T. (1979) Organization of enzymes in the shikimate pathway of Phaseolus mungo seedlings. Plant Cell Physiol. 20, 667-670 Laemmli, U.K. (1970) Cleavage of structural proteins during
83
the assembly of the head of bacteriophage T4. Nature (London) 224, 680-685 Lewendon, A., Coggins, J.R. (1983) Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli. Biochem. J. 213, 187-191 Lumsden, J., Coggins, J.R. (1977) The subunit structure of the arom multienzyme complex of Neurospora crassa. Biochem. J. 161,599-607 Patel, V.B., Giles, N.H. (1979) Purification of the arom multienzyme aggregate from Euglena gracilis. Biochim. Biophys. Acta 567, 24-34 Polley, L.D. (1978) Purification and characterisation of 3-dehydroquinate hydrolase and shikimate oxidoreductase. Biochim. Biophys. Acta 526, 259-266 Roisch, U., Lingens, F. (1980) Einflug yon N-(Phosphonomethyl)-glycin auf das Wachstum nnd auf die Enzyme der Aromatenbiosynthese von Escherichia coli. Hoppe-Seyler's Z. Physiol. Chem. 361, 1049-1058 Rubin, J.L., Gaines, C.G., Jensen, R.A. (1982) Enzymological basis for herbicidal action of glyphosate. Plant Physiol. 70, 833 839 Smith, D.D.S., Coggins, J.R. (1983) Isolation of a bifunctional domain from the pentafunctional arom enzyme complex of Neurospora crassa. Biochem. J. 213, 405-415 Steinrficken, H.C., Amrhein, N. (1980) The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic acid-3phosphate synthase. Biochem. Biophys. Res. Commun. 94, 1207-1212 Received 29 July; accepted 12 September 1983
