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
Mentions: The narrow crevice where substrates bind is likewise formed by a dimer. One subunit contributes three α-helical segments (α10, α14, and α17), which line one side of the cavity. Three non-helical segments (the β1–α2 turn, the loop between β6 and β7, and the loop following β4) are provided by the partner subunit. The active site is polar, primarily basic due to the presence of four arginine residues (32A, 106A, 409B, and 428B) and a lysine (299B). A hydrophobic patch is formed by Ile489B, Phe490B, and Leu493B (Fig. 6). Seven of these eight amino acids are strictly conserved, and one, Lys299, is conserved or replaced by arginine in more than 90% of MenD sequences.
Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.