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

• Published in: Biochemistry (Easton) 2013-07, Vol.52 (28), p.4830-4841
• Main Authors: Guo, Youzhong, Serrano, Hector, Poelarends, Gerrit J, Johnson, William H, Hackert, Marvin L, Whitman, Christian P
• Format: Article
• Language: English
• Subjects: Actinomycetales - enzymology; Amino Acid Sequence; Bacteria; Base Sequence; Carboxy-Lyases - chemistry; Carboxy-Lyases - genetics; Carboxy-Lyases - metabolism; Crystallography, X-Ray; DNA Primers; Kinetics; Molecular Sequence Data; Mutation; Protein Conformation; Pseudomonas; Pseudomonas pavonaceae; Sequence Homology, Amino Acid; Spectrometry, Mass, Electrospray Ionization
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi400567a

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 and 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. In terms of pairwise sequence, MSAD from Coryneform bacterium strain FG41 (designated FG41 MSAD) is 38% identical 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. 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 the 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 Pp 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.