Methylmalonate-semialdehyde Dehydrogenase from Bacillus subtilis

Abstract

Methylmalonate-semialdehyde dehydrogenase (MSDH) belongs to the CoA-dependent aldehyde dehydrogenase subfamily. It catalyzes the NAD-dependent oxidation of methylmalonate semialdehyde (MMSA) to propionyl-CoA via the acylation and deacylation steps. MSDH is the only member of the aldehyde dehydrogenase superfamily that catalyzes a ␤-decarboxylation process in the deacylation step. Recently, we demonstrated that the ␤-decarboxylation is rate-limiting and occurs before CoA attack on the thiopropionyl enzyme intermediate. Thus, this prevented determination of the transthioesterification kinetic parameters. Here, we have addressed two key aspects of the mechanism as follows: 1) the molecular basis for recognition of the carboxylate of MMSA; and 2) how CoA binding modulates its reactivity. We substituted two invariant arginines, Arg-124 and Arg-301, by Leu. The second-order rate constant for the acylation step for both mutants was decreased by at least 50-fold, indicating that both arginines are essential for efficient MMSA binding through interactions with the carboxylate group. To gain insight into the transthioesterification, we substituted MMSA with propionaldehyde, as both substrates lead to the same thiopropionyl enzyme intermediate. This allowed us to show the following: 1) the pK app of CoA decreases by ϳ3 units upon binding to MSDH in the deacylation step; and 2) the catalytic efficiency of the transthioesterification is increased by at least 10 4 -fold relative to a chemical model. Moreover, we observed binding of CoA to the acylation complex, supporting a CoA-binding site distinct from that of NAD(H).Methylmalonate-semialdehyde dehydrogenases (MSDHs) 4 belong to the CoA-dependent aldehyde dehydrogenases (ALDHs), which, together with their hydrolytic counterparts, make up the ALDH superfamily. ALDHs are known to be involved in many essential biological functions such as intermediary metabolism, detoxification, osmotic protection, and cellular differentiation. The enzymes catalyze the NAD(P)-dependent oxidation of a wide variety of aldehydes to their corresponding nonactivated or CoA-activated acids via a common two-step chemical mechanism. The acylation step leads to formation of a thioacyl enzyme intermediate, which then undergoes nucleophilic attack by a water or CoA molecule. Despite mechanistic similarities, previous studies have highlighted major differences in the kinetic mechanism of ALDHs depending on the nature of the deacylation step. In hydrolytic ALDHs, kinetic data support an ordered sequential mechanism in which NAD(P)H dissociates last (1-4). By contrast, CoA-dependent ALDHs exhibit a ping-pong mechanism in which the release of the reduced cofactor occurs before the transthioesterification step (5, 6). Whereas mechanistic and structural aspects have been studied extensively for hydrolytic ALDHs (7-14), considerably less is known about CoA-dependent ALDHs.MSDHs are present in a wide variety of organisms ranging from bacteria and archaea to plants and mammals where they are described to play a d...

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

“…In accordance with the ping-pong kinetic mechanism, NADH release occurs before the transthioesterification step. This rules out the requirement for a flip of the NMNH for CoA binding, an assertion that is further supported by our recent kinetic data showing that the NAD(H) and CoA binding sites do not overlap (23). An alternative explanation is that nucleophilic attack of the CoA on the decarboxylated thioacylenzyme intermediate is not possible if NMNH is present in the active site due to potential steric hindrance.…”

“…The entrance of the narrow part of the catalytic tunnel comprises, notably, the side chains of Arg-124 and Arg-301. Very recently, both residues were shown to participate not only in MMSA binding through stabilizing electrostatic interactions with the carboxylate group of the substrate but likely also in positioning MMSA efficiently relative to Cys-302 in the MSDH/NAD ϩ /MMSA ternary complex (23).…”

“…In an effort to address this gap in knowledge, our group has for several years been studying the catalytic mechanism of the methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis (22,23). This homotetrameric enzyme catalyzes the NAD ϩ -dependent oxidation of methylmalonate semialdehyde (MMSA) and malonate semialdehyde to propionyl-and acetyl-CoA, respectively, and has been reported to be involved in myo-inositol catabolism (24).…”

Metabolism of myo -Inositol by Legionella pneumophila Promotes Infection of Amoebae and Macrophages

“…Yet, MmsA uses methylmalonate as a substrate producing propionyl-CoA, while IolA uses malonate semialdehyde as a substrate producing acetylCoA. Interestingly, the B. subtilis enzyme corresponding to MmsA uses methylmalonate semialdehyde or malonate semialdehyde as a substrate for dehydrogenase reactions, producing propionylCoA or acetyl-CoA, respectively (60,61). Since L. pneumophila and B. subtilis MmsA share 42% identity on the amino acid level, the Legionella enzyme might also catalyze the dehydrogenation of methylmalonate semialdehyde as well as malonate semialdehyde.…”

Archaeal Aldehyde Dehydrogenase ST0064 fromSulfolobus tokodaii, a Paralog of Non-Phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase, Is a Succinate Semialdehyde Dehydrogenase

“…Non-phosphorylating ALDHs can be classified into two types based on CoA dependency, and the absence of the general base Glu is observed in CoA-dependent ALDHs. 24,25) Reduction of succinyl-CoA to SSA is the reverse reaction of CoA-dependent oxidation of SSA. Thus succinyl-CoA reductase might catalyze the reversible reaction with high preference for the reductive direction.…”