Mechanistic characterization of a bacterial malonate semialdehyde decarboxylase - Identification of a new activity in the tautomerase superfamily
Mechanistic characterization of a bacterial malonate semialdehyde decarboxylase - Identification of a new activity in the tautomerase superfamily
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DOI：
10.1074/jbc.m306706200
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发表时间：
2003-12-05
影响因子：
4.8
通讯作者：
Whitman, CP
中科院分区：
文献类型：
--
作者：
Poelarends, GJ;Johnson, WH;Whitman, CP
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)-X-AspX- 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 beta-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 Pro1. 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.
