Kinetic and catalytic mechanisms of the methionine-derived glucosinolate biosynthesis enzyme methylthioalkylmalate synthase.

In Brassica plants, methionine-derived aliphatic glucosinolates are chemically diverse natural products that serve as plant defense compounds, as well as molecules with dietary health-promoting effects. During their biosynthesis, methylthioalkylmalate synthase (MAMS) catalyzes the elongation reaction of the aliphatic chain. The MAMS-catalyzed condensation of 4-methylthio-2-oxobutanoic acid and acetyl-CoA generates a 2-malate derivative that either enters the pathway for the synthesis of C-glucosinolates or undergoes additional extension reactions, which lead to C- to C-glucosinolates. Recent determination of the x-ray crystal structure of MAMS from Brassica juncea (Indian mustard) provided insight on the molecular evolution of MAMS, especially substrate specificity changes, from the leucine biosynthesis enzyme α-isopropylmalate synthase but left details of the reaction mechanism unanswered. Here we use the B. juncea MAMS2A (BjMAMS2A) isoform to analyze the kinetic and catalytic mechanisms of this enzyme. Initial velocity studies indicate that MAMS follows an ordered bi bi kinetic mechanism, which based on the x-ray crystal structure, involves binding of 4-methylthio-2-oxobutanoic acid followed by acetyl-CoA. Examination of the pH-dependence of k and k/K are consistent with acid/base catalysis. Site-directed mutagenesis of three residues originally proposed to function in the reaction mechanism-Arg89 (R89A, R89K, R89Q), Glu227 (E227A, E227D, E227Q), and His388 (H388A, H388N, H388Q, H388D, and H388E)-showed that only two mutants (E227Q and H388N) retained activity. Based on available structural and biochemical data, a revised reaction mechanism for MAMS-catalyzed elongation of methionine-derived aliphatic glucosinolates is proposed, which is likely also conserved in α-isopropylmalate synthase from leucine biosynthesis in plants and microbes.