Mechanistic Characterization of a Bacterial Malonate Semialdehyde Decarboxylase

Abstract

Malonate semialdehyde decarboxylase (MSAD) has been identified as the protein encoded by the orf130 gene from Pseudomonas pavonaceae 170 on the basis of the genomic context of the gene as well as its ability to catalyze the decarboxylation of malonate semialdehyde to generate acetaldehyde. The enzyme is found in a degradative pathway for the xenobiotic nematocide trans-1,3-dichloropropene. MSAD has no sequence homology to previously characterized decarboxylases, but the presence of a conserved motif (Pro 1 -(X) 8 -Gly-Arg 11 -XAsp-X-Gln) in its N-terminal region suggested a relationship to the tautomerase superfamily. Sequence analysis identified Pro 1 and Arg 75 as potential active site residues that might be involved in the MSAD activity. The results of site-directed mutagenesis experiments confirmed the importance of these residues to activity and provided further evidence to implicate MSAD as a new member of the tautomerase superfamily. MSAD is the first identified decarboxylase in the superfamily and is possibly the first characterized member of a new and distinct family within this superfamily. Malonate semialdehyde is analogous to a ␤-keto acid, and enzymes that catalyze the decarboxylation of these acids generally utilize metal ion catalysis, a Schiff base intermediate, or polarization of the carbonyl group by hydrogen bonding and/or electrostatic interactions. A mechanistic analysis shows that the rate of the reaction is not affected by the presence of a metal ion or EDTA while the incubation of MSAD with the substrate in the presence of sodium cyanoborohydride results in the irreversible inactivation of the enzyme. The site of modification is Pro 1 . These observations are consistent with the latter two mechanisms, but do not exclude the first mechanism. Based on the sequence analysis, the outcome of the mutagenesis and mechanistic experiments, and the roles determined for Pro 1 and the conserved arginine in all tautomerase superfamily members characterized thus far, two mechanistic scenarios are proposed for the MSAD-catalyzed reaction in which Pro 1 and Arg 75 play prominent roles.The trans-1,3-dichloropropene catabolic pathway is elaborated by the soil bacterium Pseudomonas pavonaceae 170 and enables the organism to use trans-1,3-dichloropropene (Scheme 1, 1) as a sole source of carbon and energy (1). The compound, a key ingredient in the nematocides Shell D-D and Telone II, is first converted in three enzymatic steps to trans-3-chloroacrylate (2), which, in turn, is processed to acetaldehyde (4). The evolution of this pathway has been the subject of several recent studies because it may be a newly evolved pathway assembled by the bacterium in response to repeated exposure to the manmade compound, 1,3-dichloropropene (1-3).Our interest in the pathway is focused on the metabolic steps involved in the conversion of trans-3-chloroacrylate (2) to acetaldehyde (4). It has recently been determined that trans-3-chloroacrylic acid dehalogenase (CaaD) 1 converts 2 to malonate semialdehyde (3) (4, 5). Based o...

Inactivation of Malonate Semialdehyde Decarboxylase by 3-Halopropiolates: Evidence for Hydratase Activity

“…Initially, we considered the possibility that the hydratase activity might be indicative of a Schiff base mechanism (1,6). In such a mechanism, a Schiff base forms between Pro-1 and the 3-carbonyl group of 4, and the resulting iminium cation facilitates decarboxylation.…”

Crystal Structures of Native and Inactivated cis-3-Chloroacrylic Acid Dehalogenase

“…The other four families, represented by their title enzymes, are the 5-(carboxymethyl)-2-hydroxymuconate isomerase (28), 4-oxalocrotonate tautomerase (26,29), macrophage migration inhibitory factor (30), and malonate semialdehyde decarboxylase (MSAD) families (27). cis-CaaD has been classified in a separate tautomerase family, the cis-CaaD family, because of the absence of significant sequence identity with known members of the other four families (1).…”

Why are chlorinated pollutants so difficult to degrade aerobically? Redox stress limits 1,3-dichloprop-1-ene metabolism byPseudomonas pavonaceae

“…Neither DhaA nor CaaD seem to need cofactors for the dechlorination reactions that they catalyse, and the cofactors for Aldh and Adh have not been identified. In the last step of 1,3-DCP biodegradation and mineralization, 3-oxopropanoate can enter into the central metabolic pathways as acetaldehyde upon decarboxylation by a malonate semialdehyde decarboxylase [58,59].…”