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The thiamine diphosphate (ThDP) and metal-ion-dependent enzyme succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxyl ate synthase, or MenD, catalyze the Stetter-like conjugate addition of a-ketoglutarate with isochorismate to release 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate and carbon dioxide. This reaction represents the first committed step for biosynthesis of menaquinone, or vitamin K-2, a key cofactor for electron transport in bacteria and a metabolite for posttranslational modification of proteins hi mammals. The medium-resolution structure of MenD from Escherichia coli (EcMenD) in complex with its cofactor and Mn2+ has been determined two related hexagonal crystal cofactor and Mu forms. The subunit displays the typical three-domain structure observed for ThDP-dependent enzymes in which two of the domains bind and force the cofactor into a configuration that supports formation of a reactive ylide. The structures reveal a stable dieter-of-dieters association in agreement with gel filtration and analytical ultracentrifugation studies and confirm the classification of MenD in the pyruvate oxidase family of ThDP-dependent enzymes. The active site, created by contributions from a pair of subunits, is highly basic with a pronounced hydrophobic patch. These features, formed by highly conserved amino acids, match well to the chemical properties of the substrates. A model of the covalent intermediate formed after reaction with the first substrate a-ketoglutarate and with the second substrate isochorismate positioned to accept nucleophilic attack has been prepared. Thus, in addition to structural and sequence comparisons with putative MenD orthologues, provides insight into the specificity and reactivity of MenD and allows a two-stage reaction mechanism to be proposed.
- crystal structure
- enzyme mechanism
- menaquinone biosynthesis
- thiamine diphosphate cofactor
- DIPHOSPHATE-DEPENDENT ENZYMES
- THIAMIN DIPHOSPHATE
- (1R,6R)-2-SUCCINYL-6-HYDROXY-2,4-CYCLOHEXADIENE-1-CARBOXYLATE SYNTHASE
- ISOCHORISMATE SYNTHASE
- ANGSTROM RESOLUTION
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- 2 Finished
1/01/08 → 31/12/12