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

Abstract

A soil bacterium, Burkholderia sp. WS, grows on 2-chloroacrylate as the sole carbon source. To identify the enzymes metabolizing 2-chloroacrylate, we carried out comparative two-dimensional gel electrophoresis of the proteins from 2-chloroacrylate-and lactate-grown bacterial cells. As a result, we found that a protein named CAA43 was inducibly synthesized when the cells were grown on 2-chloroacrylate. The CAA43 gene was cloned and shown to encode a protein of 333 amino acid residues (M r 35,788) that shared a significant sequence similarity with NADPH-dependent quinone oxidoreductase from Escherichia coli (38.2% identity). CAA43 was overproduced in E. coli and purified to homogeneity. The purified protein catalyzed the NADPH-dependent reduction of the carbon-carbon double bond of 2-chloroacrylate to produce (S)-2-chloropropionate, which is probably further metabolized to (R)-lactate by (S)-2-haloacid dehalogenase in Burkholderia sp. WS. NADH did not serve as a reductant. Despite the sequence similarity to quinone oxidoreductases, CAA43 did not act on 1,4-benzoquinone and 1,4-naphthoquinone. 2-Chloroacrylate analogs, such as acrylate and methacrylate, were also inert as the substrates. In contrast, 2-bromoacrylate served as the substrate. Thus, we named this novel enzyme 2-haloacrylate reductase. This study revealed a new pathway for the degradation of unsaturated organohalogen compounds. It is also notable that the enzyme is useful for the production of (S)-2-chloropropionate, which is used for the industrial production of aryloxyphenoxypropionic acid herbicides.

Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains

“…WS (Kurata et al 2008;Kurata et al 2005). CAA43 catalyzes reduction of the carbon-carbon double bond of 2-chloroacrylate to (s)-2-chloropropionate coupled with NADPH oxidization to NADP + .…”

2-Haloacrylate Hydratase, a New Class of Flavoenzyme That Catalyzes the Addition of Water to the Substrate for Dehalogenation

“…For one of these bacteria, Burkholderia sp. strain WS, we previously discovered a new NADPH-dependent enzyme, 2-haloacrylate reductase (12,13). Although this enzyme does not directly remove a halogen atom from the substrate, it is supposed to participate in the metabolism of 2-CAA by catalyzing the conversion of 2-CAA into L-2-chloropropionate, which is subsequently dehalogenated by L-2-haloacid dehalogenase.…”

A Mechanistic Analysis of Enzymatic Degradation of Organohalogen Compounds

“…WS in the presence of NADPH, 2-chloropropionate was produced from 2-CAA, suggesting that an NADPH-dependent reductase acting on 2-CAA occurs in the 2-CAA-grown cells. 56,57) NADH did not replace NADPH. 2-Chloropropionate was not produced when cell-free extract from the lactate-grown cells was used.…”

“…7). 57) Two major 2-CAA-inducible proteins, named CAA43 and CAA67, were identified. Their genes were found to be located next to each other on the genomic DNA (GenBank accession no.…”

“…WS (referred to as CAA67 WS in this section to avoid confusion). 57) CAA67 YL and CAA67 WS have a nucleotide-binding motif (VXGXGXXGXXXA) in the region from position 11 to position 22, and showed weak but significant sequence similarity to several flavoproteins, including L-aspartate oxidase (e.g., NCBI accession no. YP 783305), the succinate dehydrogenase flavoprotein subunit (e.g., NCBI accession no.…”