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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Evidence that the reaction of the UDP-N-acetylglucosamine 1-carboxyvinyltransferase proceeds through the O-phosphothioketal of pyruvic acid bound to Cys115 of the enzyme.

The enzyme UDP-N-acetylglucosamine 1-carboxyvinyltransferase (enolpyruvyltransferase, EC catalyses the transfer of the intact 1-carboxyvinyl moiety of phosphoenolpyruvate to the 3'-hydroxyl group of the glucosamine moiety of UDP-(2')-N-acetylglucosamine with the concomitant release of inorganic phosphate, the first committed step in the biosynthesis of the bacterial cell wall peptidoglycan. Overexpression of the enzyme from Enterobacter cloacae in Escherichia coli allowed the isolation of large amounts of purified enzyme (approx. 900 mg/20 g fresh mass bacteria). By incubating the enzyme with 14C-labelled phosphoenolpyruvate, 32P-labelled orthophosphate and unlabelled UDP-(2')-N-acetyl-(3')-1-carboxyvinylglucosamine, we were able to isolate and characterise a reaction intermediate, covalently bound to the protein. It contains stoichiometric quantities of the C3 moiety (0.98 mol/mol) as well as of the phosphate moiety (0.95 mol/mol) of phosphoenolpyruvate relative to protein. The rapid turnover of this protein-bound intermediate in the presence of UDP-(2')-N-acetylglucosamine towards the product UDP-(2')-N-acetyl-(3')-1-carboxyvinylglucosamine suggests that the intermediate is kinetically competent. We also present evidence that the intermediate is bound as the O-phosphothioketal of pyruvic acid to Cys115 of the enzyme. This is the same Cys residue to which phosphomycin, an irreversible inhibitor of the UDP-GlcNAc carboxyvinyltransferase, binds covalently. Exchange of Cys115 for a Ser residue resulted in an inactive enzyme, demonstrating the essential role of Cys115 for the reaction. The only other enzyme known to catalyse the transfer of the intact 1-carboxyvinyl moiety of phosphoenolpyruvate to a substrate is the 3-phosphoshikimate 1-carboxyvinyltransferase (EC, the sixth enzyme of the shikimate pathway. The reaction of this synthase is known to proceed through a single, tightly but not covalently bound, tetrahedral intermediate. Even though the two enzymes share similarities in their primary amino acid sequences, their reaction mechanisms appear to be substantially different.[1]


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