Cloning, Expression, and Characterization of a cis-3-Chloroacrylic Acid Dehalogenase:  Insights into the Mechanistic, Structural, and Evolutionary Relationship between Isomer-Specific 3-Chloroacrylic Acid Dehalogenases

Abstract

The gene encoding the cis-3-chloroacrylic acid dehalogenase (cis-CaaD) from coryneform bacterium strain FG41 has been cloned and overexpressed, and the enzyme has been purified to homogeneity and subjected to kinetic and mechanistic characterization. Kinetic studies show that cis-CaaD processes cis-3-haloacrylates, but not trans-3-haloacrylates, with a turnover number of approximately 10 s(-1). The product of the reaction is malonate semialdehyde, which was confirmed by its characteristic 1H NMR spectrum. The enzyme shares low but significant sequence similarity with the previously studied trans-3-chloroacrylic acid dehalogenase (CaaD) and with other members of the 4-oxalocrotonate tautomerase (4-OT) family. While 4-OT and CaaD function as homo- and heterohexamers, respectively, cis-CaaD appears to be a homotrimeric protein as assessed by gel filtration chromatography. On the basis of the known three-dimensional structures and reaction mechanisms of CaaD and 4-OT, a sequence alignment implicated Pro-1, Arg-70, Arg-73, and Glu-114 as important active-site residues in cis-CaaD. Subsequent site-directed mutagenesis experiments confirmed these predictions. The acetylene compounds, 2-oxo-3-pentynoate and 3-bromo- and 3-chloropropiolate, were processed by cis-CaaD to products consistent with an enzyme-catalyzed hydration reaction previously established for CaaD. Hydration of 2-oxo-3-pentynoate afforded acetopyruvate, while the 3-halopropiolates became irreversible inhibitors that modified Pro-1. The results of this work revealed that cis-CaaD and CaaD have different primary and quaternary structures, and display different substrate specificity and catalytic efficiencies, but likely share a highly conserved catalytic mechanism. The mechanism may have evolved independently because sequence analysis indicates that cis-CaaD is not a 4-OT family member, but represents the first characterized member of a new family in the tautomerase superfamily that probably resulted from an independent duplication of a 4-OT-like sequence. The discovery of a fifth family of enzymes within this superfamily further demonstrates the diversity of activities and structures that can be created from 4-OT-like sequences.

Identification of a Gene Cluster EnablingLactobacillus caseiBL23 To Utilizemyo-Inositol

“…The phylogenetic analysis of iolK and homologous sequences (see Fig. S1 in the supplemental material) revealed a close relationship between the product of iolK and a malonate semialdehyde decarboxylase encoded by Pseudomonas pavonaceae (26). This result suggests that IolK also has malonate semialdehyde decarboxylase activity.…”

“…(i) In L. casei, a putative malonic semialdehyde decarboxylase gene is transcribed with the other iol genes. The gene encoding this enzyme was first described in Pseudomonas pavonaceae and was clustered in an operon involved in the degradation of the xenobiotic trans-1,3-dichloropropene (26). The presence of this gene in L. casei suggests an alternative pathway for the metabolism of malonic semialdehyde different from the classical oxidative decarboxylation catalyzed by the product of iolA (Fig.…”

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

“…The cis-and trans-3-chloroacrylic acid dehalogenases (cisCaaD and CaaD) 4 catalyze the cofactor-independent hydrolytic dehalogenation of, respectively, the cis-and trans-isomers of 3-chloroacrylic acid (1 and 2, Scheme 1) to produce malonate semialdehyde (5) and HCl (1)(2)(3). Both reactions may be initiated by the attack of water at C3 to form an enzyme-stabilized enediolate intermediate (3).…”

2-Haloacrylate Reductase, a Novel Enzyme of the Medium Chain Dehydrogenase/Reductase Superfamily That Catalyzes the Reduction of a Carbon-Carbon Double Bond of Unsaturated Organohalogen Compounds

“…Dehalogenases that catalyze the hydrolytic cleavage of the carbon-halogen bond of 3-haloacrylates to produce malonate semialdehyde occur in Pseudomonas pavanaceae 170 and a coryneform bacterium strain, FG41 (15)(16)(17). In this reaction, 3-haloacrylate is first hydrated to form an unstable halohydrin species, which subsequently degrades to produce malonate semialdehyde.…”

“…Only three enzymes catalyzing the dehalogenation of such compounds have been reported. One is corrinoid/iron-sulfur-cluster-containing reductive dehalogenase, which catalyzes the dehalogenation of tetrachloroethene and trichloroethene (14), and the others are cofactor-independent cis-and trans-3-chloroacrylate dehalogenases, which catalyze the hydration of cis-and trans-3-chloroacrylate, respectively, to produce malonate semialdehyde (15)(16)(17).…”