EPSP synthase

Figure 7 Stereoview of the structure of EPSP synthase in its open conformation.

ENZ enzyme assays, SC structural composition, MM molecular methods, IL isotopic labeling, IF isotopic fractionation, INH inhibition studies, UNK unknown, LOX lipoxogenase, EPSP synthase 5-enolpyruvylshikimate-3-phosphate, SDH shikimate dehydrogenase, PAL phenylalanine ammonium lyase, PKS polyketide synthase, NRPS nonribosomal peptide synthase 1 Gerwick 1999 2 Liu et al. 1994 3 Boonprab et al. 2003 4 Cvejic and Rohmer 1999 5 Disch et al. 1998 6 Chikaraishi et al. 2006 7 Schwender et al. 2001 8 Schwender et al. 1997 9 Mayes et al. 1993 10 Shick et al. 1999 11 Richards et al. 2006 12 Bouarab et al. 2004 13 Pelletreau et al., unpublished data 14 Dittman and Weigand 2006 15 Rein and Barrone 1999 Empty columns imply no direct evidence of these enzymes from these systems... 

Fig. 11.15 Diagram showing the relative proximity of the two ligands Glp and S3P, which form a stable ternary complex with the enzyme EPSP synthase. The distance constraints were obtained from both homonuclear and heteronuclear dipolar couplings obtained using the REDOR and DRAMA pulse sequence, together with a model showing a...

Aromatic Amino Acid Biosynthesis. The shikimate pathway is the biosynthetic route to the aromatic amino acids tryptophan, tyrosine and phenylalanine as well as a large number of secondary metabolites such as flavonoids, anthocyanins, auxins and alkaloids. One enzyme in this pathway is 5-enolpyruvyl shikimate-3-phosphate synthase (EPSP synthase) (Figure 2.9). 

A related mechanism is utilized in the biosynthesis of UDP-muramic acid (Eq. 20-6) J There is an enolpyruvoyl adduct analogous to that of EPSP synthase a proposed enolpyruvoyl-enzyme adduct with Cys 115 is not on the major path.k/1 However, this enzyme is not inhibited by glyphosate.1... 

The condensation of phospho-shikimic acid with phosphoenol pyruvate is catalyzed by the enzyme 3-enoylpyruvoylhikimate-5-phosphate synthase, or EPSP synthase for short. This reaction is specifically inhibited by the herbicide glyphosate. 

EPSP synthase catalyzes the synthesis of EPSP by an addition-elimination reaction through the tetrahedral intermediate shown in Fig. 2a. This enzyme is on the shikimate pathway for synthesis of aromatic amino acids and is the target for the important herbicide, glyphosate, which is the active ingredient in Roundup (The Scotts Company EEC, Marysville, OH). Transient-state kinetic studies led to proof of this reaction mechanism by the observation and isolation of the tetrahedral intermediate. Moreover, quantification of the rates of formation and decay of the tetrahedral intermediate established that it was tmly an intermediate species on the pathway between the substrates (S3P and PEP) and products (EPSP and Pi) of the reaction. The chemistry of this reaction is interesting in that the enzyme must first catalyze the formation of the intermediate and then catalyze its breakdown, apparently with different requirements for catalysis. Quantification of the rates of each step of this reaction in the forward and reverse directions has afforded a complete description of the free-energy profile for the reaction and allows... 

Figure 2 Intermediate in the EPSP synthase pathway, (a) The mechanism of the reaction catalyzed by EPSP synthase is shown. The reaction proceeds by an addition-elimination mechanism via a stable tetrahedral intermediate, (b) A single turnover reaction is shown in which 10- xM enzyme was mixed with 1 OO-m-M S3P and 3.5-riM radiolabeled PEP. Analysis by rapid-quench kinetic methods showed the reaction of PEP to form the intermediate, which then decayed to form EPSP in a single turnover. The smooth lines were computed from a complete model by numerical integration of the equations based on a global fit to all available data. Reproduced with permission from Reference 7.

Figure 2b shows the results of a rapid-quench single-turnover experiment performed with EPSP synthase with enzyme in excess over the radiolabeled substrate, PEP. The data show the transient formation and decay of the tetrahedral intermediate, which led to its subsequent isolation and stmcture determination. 

Anderson KS, Sikorski JA, Johnson KA. A tetrahedral intermediate in the EPSP synthase reaction observed by rapid quench kinetics. Biochemistry 1988 27 7395-7406. 

Anderson KS, Johnson KA. Kinetic and Structural Analysis of Enzyme Intermediates Lessons from EPSP Synthase. Chem. Rev. 1990 90 1131-1149. 

Anderson KS, Sammons RD, Leo GC, Sikorski JA, Benesi AJ, Johnson KA. Observation by 13C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site. Biochemistry 1990 29 1460-1465. 

Lewis J, Johnson KA, Anderson KS. The catal)4ic mechanism of EPSP synthase revisited. Biochemistry 1999 38 7372-7379... 

Dawson RMC, Elliott DC, Elliott WH, Jones KM. Data for Biochemical Research, 3rd edition. 1986. Clarendon Press, Oxford. Miller MJ, Braccolino DS, Cleary DG, Ream JE, Walker MC, Sikorski JA. EPSP synthase inhibitor design iv New aromatic substrate analogs and symmetrical inhibitors containing novel 3-phosphate mimics. Bioorg. Med. Chem. Lett. 1994 4 2605-2608. Boudreau MA, Vederas JC. Synthesis and biological evaluation of nucleoside dicarboxylates as potential mimics of nucleoside diphosphates. Org. Biomol. Chem. 2007 5 627-635. 

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