Volume 151, Issue 2 p. 443-450
Free Access

A dimeric 5-enol-pyruvyl-shikimate-3-phosphate synthase from the cyanobacterium Spirulina platensis

Giuseppe Forlani

Corresponding Author

Giuseppe Forlani

Department of Biology, University of Ferrara, via L. Borsari 46, I-44100 Ferrara, Italy

Author for correspondence: Giuseppe Forlani Tel: +39 0532 29 1311 Fax: +39 0532249761 Email:[email protected]Search for more papers by this author
Alberto Campani

Alberto Campani

Department of Biology, University of Ferrara, via L. Borsari 46, I-44100 Ferrara, Italy

Search for more papers by this author
First published: 21 December 2001
Citations: 10

Summary

  • Isolation and biochemical characterization is reported here of 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase, the enzyme that catalyses the sixth step in the common prechorismate pathway of aromatic amino acid biosynthesis and the target of the widely used herbicide glyphosate, from the cyanobacterium Spirulina platensis.

  • Homogeneous enzyme preparations were obtained by ammonium sulphate fractionation, anion-exchange and substrate-elution chromatography, and chromatofocusing. Protein characterization was carried out by conventional kinetic analysis, PAGE and gel permeation.

  • A 2800-fold purification was achieved, with a recovery of 20% of initial activity. Unusually low apparent affinities for both substrates, phosphoenolpyruvate and shikimate-3-phosphate, did not correspond to decreased glyphosate sensitivity. During SDS-PAGE, the protein migrated as a single band corresponding to a molecular mass of c. 49 kDa. The behaviour of the protein upon gel permeation chromatography under nondenaturing conditions was, however, consistent with a mass of c. 91 kDa.

  • The native enzyme appears to be homodimeric, a remarkable feature that has not been previously reported for EPSP synthases from either cyanobacteria or higher plants. The presence of mono- and dimeric EPSP synthases could represent an important tool for cyanobacterial classification.

Abbreviations

  •  
  • DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; EPSP, 5-enol-pyruvyl-shikimate-3-phosphate; PEP, phosphoenolpyruvate; S3P, shikimate-3-phosphate.
  • Introduction

    The shikimate pathway enzyme, 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19) catalyses the reversible addition of the carboxyvinyl group of phoshoenolpyruvate (PEP) to shikimate-3-phosphate (S3P), the penultimate step in the synthesis of the common intermediate in aromatic metabolism, chorismic acid (Weaver & Herrmann, 1997). The enzyme has attracted great interest because it represents the main target of the broad-spectrum, nonselective herbicide glyphosate (N-[phosphonomethyl]glycine; Kishore et al., 1992). Glyphosate may be considered an environmentally friendly pesticide, as it is rapidly and completely degraded by soil-borne microorganisms (Forlani et al., 1999). During the 1980s the inability of glyphosate to distinguish between weeds and crops severely limited its use (Dekker & Duke, 1995). However, bacterial genes encoding for glyphosate-resistant EPSP synthases were subsequently cloned, and, endowed with chloroplast transit signals, were used to transform plants (Padgette et al., 1996). Herbicide-tolerant seeds are therefore becoming available for some species of greatest agronomical value (http://www.monsanto.com/monsanto/agriculture/default.htm).

    In higher fungi, and also in several lower fungi, five of the seven reactions of the shikimate pathway, those leading from 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) to EPSP, are carried out by a single arom complex that consists of two identical pentafunctional polypeptide chains of about 170 kDa (Hawkins et al., 1993). In contrast, in bacteria and higher plants these reactions are catalysed by a bifunctional 3-dehydroquinate dehydratase/shikimate dehydrogenase (EC 4.2.1.10 and 1.1.1.25, respectively) and a number of monomeric and monofunctional proteins (Herrmann, 1995). EPSP synthase, the best studied of all the enzymes of this pathway, has been purified from a number of prokaryotic and plant species. In all cases, the estimated molecular mass of the enzyme ranged from 42 to 69 kDa for the native enzyme, and from 42 to 59 kDa under denaturing conditions (Forlani, 1992).

    Widely distributed, even in strikingly different habitats, the cyanobacteria, also referred to as blue–green algae, play a major role in biological N2 fixation. Both free-living and symbiotic strains may maintain a suitable nitrogen supply to the soil in rice fields of Far-East countries. Possible negative side-effects that the use of glyphosate may have for cyanobacteria in cultivated soils and in natural environments have not been adequately investigated to date, though a few species have been found to exhibit a remarkable natural tolerance to this herbicide (Powell et al., 1991). In the case of Anabaena variabilis, herbicide tolerance was shown to reflect an insensitive form of EPSP synthase. Under denaturing conditions the purified enzyme had an apparent molecular mass of 49 kDa and, although no data on its native composition were presented, it was considered to be a monomeric enzyme (Powell et al., 1992). An aroA homologue, coding for a putative EPSP synthase, has also been cloned from Synechocystis sp. PCC 6803. Its deduced amino acid sequence accounted for a polypeptide of 47 054 Da (Dalla Chiesa et al., 1994).

    Apart from these and a few other studies, in recent years little attention has been given to amino acid biosynthesis in cyanobacteria, and our knowledge on this fundamental aspect of cyanobacterial metabolism still relies upon early studies on partially purified preparations (Riccardi et al., 1989). This might hamper the possibility of using cyanobacteria for biotechnological purposes, for example as a nonconventional, rich source of protein for animal and, possibly, human nutrition (Ciferri, 1983) and for the large-scale production of secondary metabolites (Lagarde et al., 2000; Miyake et al., 2000).

    We previously resolved two forms of the enzyme that catalyses the first step in branched-chain amino acid synthesis, acetohydroxyacid synthase (EC 4.1.3.18), in extracts from the filamentous, nonheterocystous edible species Spirulina platensis. These appeared to be distinct isozymes, suggesting that complex regulatory mechanisms may occur in cyanobacterial nitrogen metabolism (Forlani et al., 1991). Here we report the isolation and biochemical characterization of EPSP synthase from the same strain.

    Materials and Methods

    Organism and growth conditions

    Spirulina platensis Geitler, strain C1 (Arthrospira sp. PCC 9438) was grown at 25 ± 1°C under continuous light (300 µmol m−2 s−1) on a rotary shaker (100 rpm) in 4 l Erlenmayer flasks containing 1 l of minimal medium, as previously described (Forlani et al., 1991). Subcultures were done every 2 wk by transferring 100 ml aliquots to 900 ml of fresh medium.

    Enzyme extraction and assay

    Cells in the late exponential phase of growth were harvested on filter paper by vacuum filtration, resuspended in 10 ml g−1 of ice-cold extraction buffer (50 mM Hepes-NaOH buffer, pH 7.5, containing 10% (v/v) glycerol, 2.5 mM reduced glutathione, 0.1 mM EDTA and 10 µM ammonium molybdate), and subjected to eight 20 s pulses at 80% of full power with a Branson sonifier model B12. All subsequent operations were carried out at 0–4°C. The homogenate was clarified for 15 min at 12 000 g and further centrifuged for 60 min at 100 000 g. Solid ammonium sulphate was added to the supernatant to give 70% saturation. Precipitated proteins were collected by centrifugation, resuspended in column buffer (25 mM Tris-HCl, pH 7.5, containing 0.5 mM dithiothreitol) and desalted by passage through a Bio-Gel P6DG column (Bio-Rad Laboratories, Hercules, CA, USA) equilibrated with the same buffer.

    EPSP synthase activity was measured at 35°C in the forward direction by determining release of inorganic phosphate using the malachite green dye assay method (Forlani, 1997). Briefly, in a final volume of 0.1 ml the reaction mixture contained 50 mM Hepes-NaOH, pH 7.5, 1 mM S3P, 1 mM PEP and a limiting amount of enzyme (10–25 pkat). After incubation for up to 30 min, the reaction was stopped by addition of 1 ml of the malachite green-molybdate-acid colourimetric solution followed, after exactly 1 min, by 0.1 ml of 34% (w/v) Na citrate. After 15 min at room temperature, adsorption at 660 nm was measured against blanks from which S3P had been omitted. The ammonium salt of S3P was purified from the culture broth of Klebsiella pneumoniae strain ATCC 25597 and quantified as indicated previously (Forlani et al., 1992). Protein concentration was determined by the method of Bradford (1976), using bovine serum albumin as standard.

    Enzyme purification

    EPSP synthase was purified from S. platensis by a modification of the protocol adopted for the isolation of multiple enzyme forms from cultured maize cells (Forlani et al., 1994).

    Step 1: anion exchange chromatography

    Desalted crude extracts were loaded at a constant flow rate of 60 ml h−1 onto a DEAE-Sephacel column (2.5 × 12 cm) equilibrated with column buffer. Enzyme activity was eluted with a linear gradient from 0 to 250 mM NaCl (500 ml), while collecting 5-ml fractions.

    Step 2: ammonium sulphate fractionation

    Active fractions were pooled and fractionated with ammonium sulphate. The 45–60% saturated fraction was collected by centrifugation, resuspended in 50 mM MES-NaOH buffer, pH 6.0, containing 5% (v/v) glycerol, 1.25 mM reduced glutathione, 0.1 mM EDTA and 10 µM ammonium molybdate, and desalted as above against the same buffer.

    Step 3: substrate elution chromatography

    The sample was applied at a constant flow rate of 12 ml h−1 to a cellulose phosphate column (Whatman P11, 2.5 × 4 cm) equilibrated in MES buffer. After extensive washing, proteins were eluted with a linear gradient from 0 to 1 mM S3P and PEP (200 ml), collecting 5-ml fractions. Active fractions were pooled and concentrated by centrifugation in an Ultrafree filter unit (MW cut-off 30 kDa, Millipore).

    Step 4: anion-exchange fast protein liquid chromatography

    After the pH was adjusted to 7.5, the sample was injected onto a Mono-Q 5.5 FPLC column equilibrated with column buffer. Proteins were eluted at a flow rate of 30 ml h−1 using a computer-controlled (Kontron 450) linear gradient from 0 to 400 mM NaCl (40 ml), and 0.5-ml fractions were collected.

    Step 5: chromatofocusing

    Pooled active fractions were loaded at a flow rate of 20 ml h−1 onto a Polybuffer exchanger 94 column (Sigma Chemical Co., Gallarate, Italy; 0.75 × 24 cm) equilibrated with 25 mM histidine-HCl buffer, pH 6.2. Elution proceeded with 100 ml of a 1 : 8 dilution of Polybuffer 74, while collecting 1-ml fractions. Active fractions were immediately desalted as above against column buffer, and stored at 4°C until used. Under these conditions, EPSP synthase activity was found to be stable for at least 1 wk.

    Electrophoresis

    Discontinuous SDS-PAGE was performed at 25°C by the method of Laemmli, with a 4% stacking gel and a 10% separating gel. Gels were silver-stained over a previous Coomassie Blue R250 stain (Heukeshoven & Dernick, 1985). Molecular weight markers were obtained from Sigma Chemical Co. (Product No. M3788 and M3913).

    Molecular size evaluation

    The purified protein was equilibrated with column buffer containing 0.25 M NaCl; 2-ml aliquots were loaded onto a Sephacryl S200 SF column (1.6 × 89 cm) equilibrated with the same buffer. Elution proceeded at a flow rate of 12 ml h−1, while collecting 1-ml fractions. Proteins for column calibration were supplied by Pharmacia Fine Chemicals (Pharmacia Fine Chemicals, Uppsala, Sweden, Product nos. 17–0441–01 and 17–0442–01).

    Statistical analysis

    Linear regression equations were computed by using an electronic worksheet. Data were analysed by standard statistical procedures for ANOVA and t-test. Confidence limits were computed according to Snedecor & Cochran (1967).

    Results

    Purification of S. platensis EPSP synthase

    When cyanobacterial cells were harvested during middle to late exponential growth, crude extracts contained EPSP synthase at a specific activity of 32 ± 6 pkat mg−1 protein. No significant variations were found when cells were harvested at different stages during the growth cycle (not shown). After preliminary enrichment by anion-exchange chromatography and ammonium sulphate fractionation, the active pool was subjected to substrate-elution chromatography, a high-resolution step that in several instances was found to yield homogeneous enzyme preparations (Lewendon & Coggins, 1983; Powell et al., 1992; Forlani et al., 1994). Although a 100-fold purification could be achieved by this procedure, several other proteins were still present in the eluate from the cellulose phosphate column (results not presented). This remained the case, even if the column was washed with buffer supplemented with PEP before enzyme elution, or if extracts were pretreated with a phosphocellulose column as a simple cation exchanger (Mousdale & Coggins, 1984; Forlani et al., 1994). However, the subsequent use of anion-exchange FPLC and chromatofocusing resulted in the removal of residual contaminants. The maximal specific activity observed (Table 1) reflects an enrichment of about 2 800-fold, with a yield of c. 20%. The purified enzyme was electrophoretically homogeneous: when silver stained, denaturing polyacrylamide gels showed a single, sharp band (Fig. 1). Through purification, there was no evidence for the presence of isoforms of EPSP synthase. Only a single peak of activity was evident, even when the cellulose phosphate column was eluted by a linear gradient from 0 to 0.25 mM S3P and PEP: this resulted in a broad, but symmetrical elution profile (not presented).

    Table 1. Purification of 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase from Spirulina platensis
    Protein (mg) Total activity (nkat) Specific activity (nkat mg−1) Yield (%) Purification (fold)
    Crude extract 383.6 13.34 0.035 100   1
    DEAE-Sephacel column 106.5 10.94 0.103 82.0   2.9
    45–60% ammonium sulphate fraction 30.19 8.03 0.266 60.2   7.6
    Cellulose phosphate column 0.202 4.31 21.34 32.3 610
    Mono-Q column 0.055 3.51 63.8 26.3 1823
    Polybuffer exchanger 94 column 0.0283 2.77 97.8 20.8 2794
    • Results are for a typical purification starting from 12 g (fresh weight) of cyanobacterial cells.
    Details are in the caption following the image

    Electrophoresis of 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase from Spirulina platensis under denaturing conditions (SDS-PAGE). Approx. 0.1 µg of the purified protein was loaded in lane 2; the molecular masses of protein standards (lanes 1 and 3) run on the same gel are indicated. The gel was silver-stained.

    Characterization of structural and functional properties of the cyanobacterial enzyme

    The purified protein was thoroughly characterized with respect to physical, kinetic and biochemical properties (Table 2). Temperature optimum and stability were almost identical, suggesting that the catalytically active conformation could be as stable as the inactive conformation. There were no pronounced effects of pH on EPSP synthase activity, which showed a broad optimum for catalysis over the pH range 6.7–8.6. A noteworthy acidic isoelectric point, similar to that found for the eubacterial enzyme (Steinrücken & Amrhein, 1984), may explain the difficulties met with cation-exchange chromatography. The activation energy was similar to that of EPSP synthase purified from other sources. However, the catalytic constant was more than one order of magnitude lower than that of the plant enzyme (Forlani, 1997), though consistent with that of the pure enzyme from all of the other cyanobacterial species showed to date (Powell et al., 1992). Kinetic analysis gave unusually high values for the apparent affinity constants for both S3P and PEP (Table 2). Since it was possible, even if unlikely, that substrate elution chromatography may result in a residual presence of the two compounds tightly bound to the enzyme, kinetic experiments were repeated with the enzyme partially purified by means of an alternative procedure in which the cellulose phosphate step was omitted. Very similar values were obtained for the apparent Km values (not shown). Because high affinity constants for PEP have previously been shown always to correlate with natural (Forlani et al., 1992; Padgette et al., 1996) or selected (Kishore et al., 1992) tolerance to the phosphonate herbicide glyphosate, enzyme activity was studied in the presence of increasing concentrations of the inhibitor. As expected, the inhibition was of competitive type with respect to PEP, and uncompetitive with respect to S3P (Fig. 2). The calculated Ki values (Table 2) were significantly higher than those for the plant enzyme (Forlani, 1992, 1997), but only slightly higher than those for other microbial EPSP synthases (Powell et al., 1992, and the references therein). In the presence of saturating substrate, the concentration of glyphosate causing 50%-inhibition of the catalytic rate (28 ± 2 µM) places the S. platensis enzyme into the most sensitive of the three categories characterized among bacterial EPSP synthases (Schulz et al., 1985).

    Table 2. Structural and functional properties of EPSP synthase from S. platensis
    Molecular mass Denatureda 49.1 ± 3.0 kDa
    Nativeb 91.4 ± 2.2 kDa
    Isoelectric pointc 4.35 ± 0.06
    Activation energyd 56.1 kJ mol−1
    Catalytic constante 4.8 s−1
    Km (app) For PEPf 345 ± 25 µM
    For S3Pg 303 ± 4 µM
    Ki for glyphosate With respect to PEPh 4.5 ± 0.1 µM
    With respect to S3Pi 31.4 ± 2.3 µM
    Temperature optimumj 47.5°C
    Temperature stabilityk 46°C
    pH optimuml 7.2, 8.0
    • a Mean values determined from relative mobilities during SDS-PAGE; see Fig. 3(a). bMean values determined from elution volumes upon gel permeation chromatography; see Fig. 3(b). cValue estimated from the pH profile in the eluate from the Polybuffer exchanger 94 column. dThe result was obtained by replotting data (Ferguson plot) from two independent experiments in which the catalytic rate was measured as a function of temperature. eKcat was calculated from specific activity value of the purified protein on the basis of its molecular mass as estimated by SDS-PAGE. fThe second nonvariable substrate was fixed at 1 mM. Mean ± SD of four replications in which concentration for the variable substrate ranged from 60 to 400 µM. gThe second nonvariable substrate was fixed at 1 mM. Mean ± SD of three replications in which the concentration for the variable substrate ranged from 50 to 500 µM. hCompetitive inhibition (see Fig. 2a), slope replots. Mean ± SD of two independent experiments. Each time various glyphosate levels were tested (0, 5, 10 and 15 µM) at varying PEP concentrations. iUncompetitive inhibition (Fig. 2b), intercept replots. Mean value from two experiments, each by evaluating the effect of glyphosate levels from 0 to 100 µM at different S3P concentration (125, 250, 350, 500 µM). jOptimal temperature was defined as the temperature at which maximum enzyme activity was obtained following a 4-min-incubation. The experiment was repeated twice. kActivity of the purified protein was measured at 35°C following treatment for 10 min at various temperatures (from 25 to 60°C); enzyme stability was defined as the ability to retain more than 90% of the activity of untreated controls. lEnzyme activity was measured as a function of pH in the presence of an equimolar mixture of MES-HEPES-AMPSO buffers (0.066 M each), brought to different pH values with NaOH.
    Details are in the caption following the image

    Kinetic analysis of 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase inhibition by glyphosate. Enzyme activity was evaluated at varying substrate concentrations in the presence of increasing levels of the herbicide. The second invariable substrate was fixed at 1 mM. Parallel lines in Lineweaver-Burk plots show an inhibition of uncompetitive type with respect to S3P (panel b), while lines converging to the x-axis account for competitive inhibition with respect to PEP (panel a). a.u., arbitrary units. (a) circles, 0 mM; triangles, 5 mM; diamonds, 10 mM. (b) circles, 125 µM; triangles, 250 µM; diamonds, 500 µM

    Biochemical evidence for a homodimeric composition of the purified protein

    When S. platensis EPSP synthase was subjected to PAGE under denaturing conditions, it migrated to a position that corresponds to a molecular mass of 49 kDa (Fig. 3a), a value consistent with those estimated for A. variabilis (49 kDa; Powell et al., 1992) and deduced for the Synechocystis polypeptide (47 kDa, Dalla Chiesa et al., 1994). However, the behaviour of the enzyme upon gel permeation chromatography suggested a native molecular mass of about 91 kDa (Fig. 3b). Gel filtration chromatography in the presence of high ionic strength (up to 1 M NaCl) did not change the elution properties, thus making the occurrence of protein aggregation during the run unlikely. In addition, denaturing gels at higher acrylamide concentration did not reveal any other low-molecular-weight polypeptide. Under the same experimental conditions, EPSP synthase partially purified from Nostoc sp. strain Cc2, showed a retention pattern during gel filtration consistent with a native mass of about 38 kDa (A. Campani and G. Forlani, unpublished), whilst the two enzyme forms previously purified from cultured cells of maize showed molecular masses evaluated by gel filtration that differed only slightly from those estimated by SDS-PAGE (Forlani et al., 1994). This would appear to eliminate the possibility that methodological artifacts explain the larger molecular mass estimated for EPSP synthase from S. platensis during gel filtration. Moreover, purified EPSP synthase from S. platensis was completely devoid of shikimate dehydrogenase activity (data not shown), suggesting that even the occurrence of a multifunctional arom complex similar to those present in fungi (Hawkins et al., 1993) is unlikely. Thus the S. platensis enzyme appears to be a monofunctional homodimeric protein.

    Details are in the caption following the image

    Molecular mass of 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase from Spirulina platensis (triangles), estimated by SDS-PAGE (panel a) and gel permeation chromatography (panel b). In the former case mean values of relative mobilities from four independent runs were plotted. In the latter, retention patterns obtained upon gel filtration on a Sephacryl S200 column were used to correlate elution volumes with the logarithm of molecular weight, as described in Pharmacia technical bulletin 11-B-033-04 (1985); mean values over three replications were used. The corresponding values obtained with protein standards (circles) are also indicated.

    Discussion

    Except for the glyphosate-resistant enzyme isolated from A. variabilis (Powell et al., 1992), this is the first report describing the purification and characterization of EPSP synthase from a cyanobacterium. Possibly resulting from differences in substrate binding, the conventional high-resolving step of substrate elution chromatography did not succeed in yielding homogeneous preparations of EPSP synthase from Spirulina platensis, and had to be followed by further enrichment steps. The elution by PEP alone of at least part of the cellulose phosphate-bound enzyme activity seems not to be consistent with an ordered sequential mechanism whereby S3P must bind before PEP, as has previously been proposed on the basis of a kinetic analysis of the inhibition of EPSP synthase by glyphosate (Steinrücken & Amrhein, 1984; Ream et al., 1988). However, recent findings have suggested that glyphosate may interact with the enzyme in a different way from that assumed in the earlier studies (Sammons et al., 1995).

    The properties of purified EPSP synthase from S. platensis were, on the whole, consistent with those of the monofunctional enzyme previously detected in bacteria and higher plants. However, unusually low affinities for both substrates were found. Apparent affinity constants (345 and 303 µM for PEP and S3P, respectively) were 10–100-fold higher than those for the enzyme from A. variabilis (36 and 6 µM; Powell et al., 1992) or from other sources, which generally ranged from 1 to 30 µM (Forlani, 1992). This low affinity for PEP, however, did not correspond to a low sensitivity to glyphosate. Even if, as expected, the inhibition brought about by the herbicide was competitive with respect to the substrate (Fig. 2), the calculated Ki value was relatively low. Due to its independency from absolute affinity values, which in plants are about one order of magnitude lower than in bacteria (Herrmann, 1995), the Ki : Km ratio constitutes a better parameter from which to estimate glyphosate tolerance. In sensitive species Ki : Km ranges from 0.01 to 0.1, while for both natural and selected resistant enzymes it is greater than 10 (Kishore et al., 1992; Powell et al., 1992), indicating that glyphosate binding is affected to a greater extent than PEP binding. In the case of EPSP synthase from S. platensis, Ki : Km had a value of 0.013. Biochemical evidence supporting glyphosate as a transition state inhibitor (summarized in Sikorski & Gruys, 1997), as well as the fact that EPSP synthases with a decreased affinity for glyphosate also exhibited higher values of the affinity constant for PEP (e.g. Forlani et al., 1992), led originally to a general consensus that there should be substantial overlap between the binding domains for PEP and glyphosate. In more recent years, however, convincing evidence has been shown for the nonequivalency of PEP and herbicide binding (McDowell et al., 1996; Sikorski & Gruys, 1997). Our data seem consistent with such a lack of direct correlation between glyphosate inhibition and potency or loss of catalytic efficiency (Herrmann & Weaver, 1999). Essentially therefore the purified protein has simply to be regarded to as a poorly efficient enzyme. This evaluation is strengthened by the results obtained under substrate-saturating conditions. The turnover number (about 5 EPSP molecules produced s−1) is significantly lower than that of the eubacterial (c. 20 s−1; Lewendon & Coggins, 1983; Steinrücken & Amrhein, 1984) and plant (about 100 s−1; Forlani, 1997) enzymes, even if consistent with that of EPSP synthase from A. variabilis (about 1 s−1; Powell et al., 1992). So during evolutionary radiation, selection appears to have increased the catalytic efficiency of EPSP synthase, possibly in connection with the need to produce the huge amount of secondary metabolites that in higher plants derive from aromatic amino acids (Haslam, 1993). On the other hand, such low affinities could provide the cyanobacterial cell with a simple, although expensive, way to regulate carbon flow through metabolism by a simple overflow mechanism driven by the activity of the first enzyme in the pathway, DAHP synthase (EC 2.1.2.15).

    In agreement with data from all other sources except fungi, in which enzyme activity is part of a pentafunctional arom complex that catalyses the direct conversion of DAHP to EPSP (Hawkins et al., 1993), EPSP synthase from S. platensis is made up of a single polypeptide of molecular mass 49 kDa (Fig. 1). Interestingly, however, the retention pattern of this protein during gel permeation chromatography was consistent with a significantly larger mass (Fig. 3b). Although gel filtration more properly measures hydrodynamic properties such as the Stokes’ radius (an estimate of the mean size of the protein along its three dimensions), it is commonly used to estimate the molecular mass of enzymes, even in crude preparations. The discrepancy between the results obtained with the two methods is too high to rely entirely upon experimental inaccuracy, and several checks were done to rule out possible errors. It is concluded therefore that the native holoenzyme is most likely composed of two identical subunits, a feature that has never been described to date for EPSP synthase from either cyanobacteria or higher plants, even if not unusual for other enzymes in the shikimate pathway. For example, a homodimeric structure was reported for both DAHP synthase (Herrmann, 1995) and chorismate synthase (EC 4.6.1.4; Henstrand et al., 1995) in plants. The occurrence of multimeric forms of EPSP synthase has previously been demonstrated only in the case of the Gram-positive bacterium Bacillus subtilis. Steady-state kinetic analysis of this enzyme revealed allosteric behaviour, with both substrates showing multiple interaction sites. Moreover, two sites were found for PEP binding, catalytic and noncatalytic. Glyphosate competed for binding at the catalytic site and did not interact at the secondary site. In the absence of ammonium ions the herbicide increased cooperativity of PEP binding and favoured dimerization of the enzyme through an interaction between PEP-binding sites (Majumder et al., 1995). However, the characterization of EPSP synthase from another Gram-positive bacterium, Streptococcus pneumoniae, recently ruled out glyphosate-binding synergy mechanisms that may derive from, or are influenced by, changes in oligomerization of the target enzyme. Rather, the data suggested an allosteric mechanism involving changes in tertiary structure (Du et al., 2000).

    The distribution of a dimeric enzyme among cyanobacterial species is worthy of further investigation. Our preliminary results have confirmed the presence of dimeric EPSP synthases in other cyanobacteria, though in Nostoc sp. a monomeric enzyme was detected (A. Campani & G. Forlani, unpublished). Although traditional classification suggested the existence of a huge number of cyanobacterial species, in recent years the adoption of molecular methods has yielded quite different results. Thus, one genotype may have more than one morphotype, and several strains previously assigned to the same genus should be reclassified in independent taxonomic units. However, due to the strong dependence of analysis output upon the tree inference method employed, strikingly different distance trees were proposed. Data on the presence of mono- and dimeric EPSP synthases could thus represent a useful tool to prove the significance of phylogenetic trees based upon 16S rRNA sequence analysis.

    Acknowledgements

    We wish to thank Dr Daniela Sirotti for skilful technical assistance, and Prof Erik Nielsen and Anna Maria Sanangelantoni for helpful advice.