Mechanistic characterization of the MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis

Homotetrameric MSDH (methylinalonate semialdehyde dehydrogenase) from Bacillus subtilis catalyses the NAD-dependent oxidation of MMSA (methylinalonate semialdehyde) and MSA (malonate semialdehyde) into PPCoA (propionyl-CoA) and acetyl-CoA respectively via a two-step mechanism. In the present study, a detailed mechanistic characterization of. the MSDH-catalysed reaction has been carried out. The results suggest that NAD binding elicits a structural imprinting of the apoenzyme, which explains the marked lag-phase observed in the activity assay. The enzyme also exhibits a half-of-the-sites reactivity, with two subunits being active per tetramer. This result correlates well with the presence of two populations of catalytic Cys(302) in both the apo- and holo-enzymes. Binding of NAD causes a decrease in reactivity of the two Cys(302) residues belonging to the two active subunits and a pK(app) shift from approx. 8.8 to 8.0. A study of the rate of acylation as a function of pH revealed a decrease in the pK(app) of the two active Cys(302) residues to approx. 5.5. Taken together, these results support a sequential Cys(302) activation process with a pK(app) shift from approx. 8.8 in the apo-form to 8.0 in the binary complex and finally to approx. 5.5 in the ternary complex. The rate-limiting step is associated with the beta-decarboxylation process which occurs on the thioacylenzyme intermediate after NADH release and before transthioesterification. These data also indicate that bicarbonate, the formation of which is enzymecatalysed, is the end-product of the reaction.