Kinetic, Mutational, and Structural Analysis of Malonate Semialdehyde Decarboxylase from Coryneform Bacterium Strain FG41: Mechanistic Implications for the Decarboxylase and Hydratase Activities

Abstract

Malonate semialdehyde decarboxylase from Pseudomonas pavonaceae 170 (designated Pp MSAD) is in a bacterial catabolic pathway for the nematicide 1,3-dichloropropene. MSAD has two known activities: it catalyzes the metal-ion independent decarboxylation of malonate semialdehyde to produce acetaldehyde and carbon dioxide, as well as a low-level hydration of 2-oxo-3-pentynoate to yield acetopyruvate. The latter activity is not known to be biologically relevant. Previous studies identified Pro-1, Asp-37, and a pair of arginines (Arg-73 and Arg-75) as critical residues in these activities. MSAD from Coryneform bacterium strain FG41 (designated FG41 MSAD) shares 38% pairwise sequence identity with the Pseudomonas enzyme including Pro-1 and Asp-37. However, Gln-73 replaces Arg-73, and the second arginine is shifted to Arg-76 by the insertion of a glycine. In order to determine how these changes relate to the activities of FG41 MSAD, the gene was cloned and the enzyme expressed and characterized. The enzyme has a comparable decarboxylase activity, but a significantly reduced hydratase activity. Mutagenesis along with crystal structures of the native enzyme (2.0 Å resolution) and the enzyme modified by a 3-oxopropanoate moiety (resulting from the incubation of enzyme and 3-bromopropiolate) (2.2 Å resolution) provided a structural basis. The roles of Pro-1 and Asp-37 are likely the same as those proposed for MSAD. However, the side chains of Thr-72, Gln-73, and Tyr-123 replace those of Arg-73 and Arg-75 in the mechanism and play a role in binding and catalysis. The structures also show that Arg-76 is likely too distant to play a direct role in the mechanism. FG41 MSAD is the second functionally annotated homologue in the MSAD family of the tautomerase superfamily and could represent a new subfamily.

Reactions of Cg10062, a cis-3-Chloroacrylic Acid Dehalogenase Homologue, with Acetylene and Allene Substrates: Evidence for a Hydration-Dependent Decarboxylation

“…10,20 The decarboxylation of exogenous 4 by Cg10062 and the mutants was monitored by the coupled assay (above) without MSAD and by 1 H NMR spectroscopy.…”

Identification and characterization of new family members in the tautomerase superfamily: Analysis and implications

“…The mechanisms for the decarboxylation of 7 and the hydration of 3 parallel those proposed for the Pp MSAD in Schemes 8 and 9, respectively, where the arginine pair (in Pp MSAD) is replaced by Thr-72, Gln-73, and Tyr-123 (Scheme 10) [43,45]. Hence, the major changes in the mechanisms involve the carboxylate end of both molecules ( 3 and 7 ).…”

“…MSAD has two characterized activities: it catalyzes the metal-ion independent decarboxylation of 7 to produce acetaldehyde and carbon dioxide, as well as the hydration of 3 to yield 4 ( k cat / K m ~ 600–1130 M −1 s −1 ) (Table 2) [28,43]. The key residues for these activities (based primarily on crystallographic and mutagenesis studies) are Pro-1, Asp-37, and a pair of arginines (Arg-73 and Arg-75) [44,45].…”

“…MSAD from Coryneform bacterium strain FG41 (designated FG41 MSAD) is also part of a pathway for the degradation of 5 [43]. The enzyme has 38% sequence identity and 65% sequence similarity with the Pp MSAD.…”

“…A mutagenic and crystallographic analysis suggested mechanisms for both activities and uncovered differences between the FG41 MSAD and the Pp MSAD [43]. Mutagenesis experiments show that Pro-1, Asp-37, and Arg-76 are essential for the decarboxylase and hydratase activities.…”