Structure and reactivity of Bacillus subtilis MenD catalyzing the first committed step in menaquinone biosynthesis.
Bottom Line: Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of alpha-ketoglutarate.Mutagenesis of Phe490 and Ile489 has the most profound influence on catalytic efficiency, indicating that these two residues are important for binding of isochorismate and for stabilizing the cofactor position.These data allow for a detailed description of the structure-reactivity relationship that governs MenD function and refinement of the model for the catalytic intermediate that supports the Stetter-like conjugate addition.
Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.Show MeSH
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Mentions: The structure of MenD and our model (Fig. 6) of the activated intermediate–isochorismate complex suggested that the actual enzyme mechanism is driven by the chemical properties of the cofactor ThDP. A two-stage mechanism that corresponds to reactions with each of the substrates can be proposed (Fig. 7).15 The absence of any residue that can act as a general acid/base indicates that the cofactor N4′ is a critical component of the mechanism. Such an observation is consistent with structural and mechanistic studies of another ThDP-dependent enzyme, N2-(2-carboxyethyl)arginine synthase.23
Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.