Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli.

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.     

Crystallization of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli

Crystals of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli have been grown out of ammonium sulfate by the hanging drop method of vapor diffusion. The crystals belong to the hexagonal space group P6₁22 or P6₅22, with $\text{a = 124}\text{A}\text{?}\text{and}\text{c = 381}\text{A}\text{?}$, and diffract to 3.8 Å resolution.     

Characterization of 5-enolpyruvylshikimate 3-phosphate synthase gene from Camptotheca acuminata

5-enolpyruvylshikimate 3-phosphate synthase (EPSPS; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC 2.5.1.19) is a critical enzyme in the shikimate pathway. The full-length EPSPS cDNA sequence (CaEPSPS, GenBank accession number: AY639815) was cloned and characterized for the first time from woody plant, Camptotheca acuminata, using rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of CaEPSPS was 1778 bp containing a 1557 bp ORF (open reading frame) encoding a polypeptide of 519 amino acids with a calculated molecular mass of 55.6 kDa and an isoelectric point of 8.22. Comparative and bioinformatic analyses revealed that CaEPSPS showed extensive homology with EPSPSs from other plant species. CaEPSPS contained two highly conserved motifs owned by plant and most bacteria EPSPSs in its N-terminal region. Phylogenetic analysis revealed that CaEPSPS belonged to dicotyledonous plant EPSPS group. Tissue expression pattern analysis indicated that CaEPSPS was constitutively expressed in leaves, stems and roots, with the lower expression being found in roots. The coding sequence of CaEPSPS gene was successfully subcloned in a plasmid-Escherichia coli system (pET-32a), and the cells containing the plasmid carrying the CaEPSPS gene exhibited enhanced tolerance to herbicide glyphosate, compared to the control.     

Purification and properties of 5-enolpyruvylshikimate 3-phosphate synthase from seedlings of Pisum sativum L.

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, ion-exchange 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.Abbreviations DEAE diethylaminoethyl - EPSP 5-enolpyruvylshikimate 3-phosphate     

The purification of 5-enolpyruvylshikimate 3-phosphate synthase from an overproducing strain of Escherichia coli

The Escherichia coli aroA gene which codes for the enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSP synthase) has been cloned from the lambda-transducing bacteriophage lambda pserC. The gene has been located on a 4.7 kilobase pair PstI DNA fragment which has been inserted into the multiple copy plasmid pAT153. E. coli cells transformed with this recombinant plasmid overproduce EPSP synthase 100-fold. A simple method for the purification of homogeneous enzyme in milligram quantities has been devised. The resulting enzyme is indistinguishable from enzyme isolated from untransformed E. coli.     

Purification and properties of a glyphosate-tolerant 5-enolpyruvylshikimate 3-phosphate synthase from the cyanobacterium Anabaena variabilis

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.9) from the glyphosate-tolerant cyanobacterium Anabaena variabilis (ATCC 29413) was purified to homogeneity. The enzyme had a similar relative molecular mass to other EPSP synthases and showed similar kinetic properties except for a greatly elevated K _i for the herbicide glyphosate (approximately ten times higher than that of enzymes from other sources). With whole cells, the monoisopropylamine salt of glyphosate was more toxic than the free acid but the effects of the free acid and monoisopropylamine salt on purified EPSP synthase were identical.Abbreviations EPSP 5-enolpyruvylshikimate 3-phosphate - M_r relative molecular mass - PEP phosphoenolpyruvate - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - S3P shikimate 3-phosphate The funding of this work by the Agricultural and Food Research Council and the University of Dundee Research Initiatives Programme is gratefully acknowledged.     

Characterization of Streptococcus pneumoniae 5-enolpyruvylshikimate 3-phosphate synthase and its activation by univalent cations.

The aroA gene (Escherichia coli nomenclature) encoding 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the gram-positive pathogen Streptococcus pneumoniae has been identified, cloned and overexpressed in E. coli, and the enzyme purified to homogeneity. It was shown to catalyze a reversible conversion of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to EPSP and inorganic phosphate. Activation by univalent cations was observed in the forward reaction, with NH+4, Rb+ and K+ exerting the greatest effects. Km(PEP) was lowered by increasing [NH+4] and [K+], whereas Km(S3P) rose with increasing [K+], but fell with increasing [NH+4]. Increasing [NH+4] and [K+] resulted in an overall increase in kcat. Glyphosate (GLP) was found to be a competitive inhibitor with PEP, but the potency of inhibition was profoundly affected by [NH+4] and [K+]. For example, increasing [NH+4] and [K+] reduced Ki(GLP versus PEP) up to 600-fold. In the reverse reaction, the enzyme catalysis was less sensitive to univalent cations. Our analysis included univalent cation concentrations comparable with those found in bacterial cells. Therefore, the observed effects of these metal ions are more likely to reflect the physiological behavior of EPSP synthase and also add to our understanding of how to inhibit this enzyme in the host organism. As there is a much evidence to suggest that EPSP synthase is essential for bacterial survival, its discovery in the serious gram-positive pathogen S. pneumoniae and its inhibition by GLP indicate its potential as a broad-spectrum antibacterial target.     

Molecular basis for the glyphosate-insensitivity of the reaction of 5-enolpyruvylshikimate 3-phosphate synthase with shikimate

The shikimate pathway enzyme 5-enolpyruvyl shikimate-3-phosphate synthase (EPSP synthase) has received attention in the past because it is the target of the broad-spectrum herbicide glyphosate. The natural substrate of EPSP synthase is shikimate-3-phosphate. However, this enzyme can also utilize shikimate as substrate. Remarkably, this reaction is insensitive to inhibition by glyphosate. Crystallographic analysis of EPSP synthase from Escherichia coli, in complex with shikimate/glyphosate at 1.5 Angstroms resolution, revealed that binding of shikimate induces changes around the backbone of the active site, which in turn impact the efficient binding of glyphosate. The implications from these findings with respect to the design of novel glyphosate-insensitive EPSP synthase enzymes are discussed.     

Pre-steady-state reaction of 5-aminolevulinate synthase. Evidence for a rate-determining product release

5-Aminolevulinate synthase (ALAS) is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and the alpha-subclass of purple bacteria. The pyridoxal 5'-phosphate cofactor at the active site undergoes changes in absorptive properties during substrate binding and catalysis that have allowed us to study the kinetics of these reactions spectroscopically. Rapid scanning stopped-flow experiments of murine erythroid 5-aminolevulinate synthase demonstrate that reaction with glycine plus succinyl-CoA results in a pre-steady-state burst of quinonoid intermediate formation. Thus, a step following binding of substrates and initial quinonoid intermediate formation is rate-determining. The steady-state spectrum of the enzyme is similar to that formed in the presence of 5-aminolevulinate, suggesting that release of this product limits the overall rate. Reaction of either glycine or 5-aminolevulinate with ALAS is slow (kf = 0.15 s-1) and approximates kcat. The rate constant for reaction with glycine is increased at least 90-fold in the presence of succinyl-CoA and most likely represents a slow conformational change of the enzyme that is accelerated by succinyl-CoA. The slow rate of reaction of 5-aminolevulinate with ALAS is 5-aminolevulinate-independent, suggesting that it also represents a slow isomerization of the enzyme. Reaction of succinyl-CoA with the enzyme-glycine complex to form a quinonoid intermediate is a biphasic process and may be irreversible. Taken together, the data suggest that turnover is limited by release of 5-aminolevulinate or a conformational change associated with 5-aminolevulinate release.     

Identification of a glyphosate-resistant mutant of rice 5-enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy

5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway and is targeted by the wide-spectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C(317)-->T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (Ki and Km) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops.     

Chorismate mutase and 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the methylotrophic actinomycete Amycolatopsis methanolica.

Chorismate mutase (CM) and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS) are key regulatory enzymes in L-Phe and L-Tyr biosynthesis in Amycolatopsis methanolica. At least two CM proteins, CMIa and CMIb, are required for the single chorismate mutase activity in the wild type. Component CMIa (a homodimeric protein with 16-kDa subunits) was purified to homogeneity (2,717-fold) and kinetically characterized. The partially purified CMIb preparation obtained also contained the single DS (DSI) activity detectable in the wild type. The activities of CMIa and CMIb were inhibited by both L-Phe and L-Tyr. DSI activity was inhibited by L-Trp, L-Phe, and L-Tyr. A leaky L-Phe-requiring auxotroph, mutant strain GH141, grown under L-Phe limitation, possessed additional DS (DSII) and CM (CMII) activities. Synthesis of both CMII and DSII was repressed by L-Phe. An ortho-DL-fluorophenylalanine-resistant mutant of the wild type (strain oFPHE83) that had lost the sensitivity of DSII and CMII synthesis to L-Phe repression was isolated. DSII was partially purified (a 42-kDa protein); its activity was strongly inhibited by L-Tyr. CMII was purified to homogeneity (93.6 fold) and characterized as a homodimeric protein with 16-kDa subunits, completely insensitive to feedback inhibition by L-Phe and L-Tyr. The activity of CMII was activated by CMIb; the activity of CMII plus CMIb was again inhibited by L-Phe and L-Tyr. A tightly blocked L-Phe- plus L-Tyr-requiring derivative of mutant strain GH141, GH141-19, that had lost both CMIa and CMII activities was isolated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and overexpression in soluble form of functional shikimate kinase and 5-enolpyruvylshikimate 3-phosphate synthase enzymes from Mycobacterium tuberculosis

Tuberculosis (TB) resurged in the late 1980s and an estimated 1.87 million people died of TB in 1997. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multidrug-resistant strains have created a need to develop new antimycobacterial agents. The existence of a shikimate pathway has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis aroK-encoded shikimate kinase and aroA-encoded 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase were cloned and the enzymes overexpressed in soluble form. Overexpression was achieved without isopropyl beta-d-thiogalactoside induction, and cells grown to stationary phase yielded approximately 30% of target proteins to total soluble cell proteins. Enzyme activity measurements using coupled assays demonstrated that there was a 328-fold increase in specific activity for shikimate kinase and 101-fold increase for EPSP synthase.     

Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0 ×10⁴ clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indicajaponica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.     

Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0×104 clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.     

Spectroscopic and kinetic characterization of the bifunctional chorismate synthase from Neurospora crassa: evidence for a common binding site for 5-enolpyruvylshikimate 3-phosphate and NADPH

Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN:NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed "bifunctional." We have cloned chorismate synthase from the common bread mold Neurospora crassa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant N. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced flavin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity.     

3-Deoxy-D-manno-octulosonate-8-phosphate synthase from Escherichia coli. Model of binding of phosphoenolpyruvate and D-arabinose-5-phosphate

The crystal structure of 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli was determined by molecular replacement using coordinates given to us by Radaev and co-workers prior to publication. The KDOPS crystals reported by Radaev et al. were grown in the presence of 1.4 M (NH(4))(2)SO(4) and 0.4 M (K/H)(3)PO(4). They are in the cubic space group I23 (a=228.6 A) with a tetramer in the asymmetric unit; the structure has been refined with data to 2.4 A. Our crystals of E. coli KDOPS, grown in 24 % (w/v) polyethylene glycol (PEG) 1500 in the presence of the substrates, 2-phosphoenolpyruvate (PEP) and d-arabinose-5-phosphate (A5P), are also in space group I23 (a=118.2 A), with one subunit in the asymmetric unit.The medium of crystallization, 1.8 M SO(4)/PO(4) versus 24 % PEG, does not significantly affect the conformation of KDOPS. The inter-monomer contacts in both structures are the same. The beta(8)/alpha(8) loop (residues 246 to 251) situated near the entrance to the active site is not seen in the 229 A structure but can be traced in the 118 A structure.Most significantly, Radaev et al. interpreted two SO(4)/PO(4) sites in the 229 A structure as marking the phosphate positions of the substrates, PEP and A5P, after the precedent of DAHPS. In the 118 A structure the inner of these two SO(4)/PO(4) peaks is present at the same position as in the 229 A structure of KDOPS. The outer phosphate peak in the 118 A KDOPS is 3.7 A from the outer SO(4)/PO(4) peak in the 229 A structure and is within hydrogen bonding distance of Arg63 of the same subunit and Arg120 of another subunit. Based on the precedent of the d-erythrose-4-phosphate (E4P) modeled in the active site of DAHPS, we have modeled PEP and A5P in KDOPS and compared the coordination of PEP and A5P in KDOPS with that of PEP and E4P in DAHPS.     

5-enolpyruvylshikimate 3-phosphate synthase, the target enzyme of the herbicide glyphosate, is synthesized as a precursor in a higher plant

Cell cultures of Corydalis sempervirens adapted to growth in the presence of 5 millimolar glyphosate overproduce the herbicide's target enzyme, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, 30- to 40-fold. In vitro translation of total RNA and poly(A)-RNA coupled with immunoprecipitation showed that the protein is synthesized as a precursor of relative molecular weight (M_r) 53900 ± 900 as compared to M_r 45500 ± 1000 of the mature enzyme. Translatable activity of mRNA for EPSP-synthase in glyphosate-adapted cultures is tenfold higher than in nonadapted cultures.