2-Haloacrylate Hydratase, a New Class of Flavoenzyme That Catalyzes the Addition of Water to the Substrate for Dehalogenation
2010
Mowafy, Amr M. | Kurihara, Tatsuo | Kurata, Atsushi | Uemura, Tadashi | Esaki, Nobuyoshi
Enzymes catalyzing the conversion of organohalogen compounds are useful in the chemical industry and environmental technology. Here we report the occurrence of a new reduced flavin adenine dinucleotide (FAD) (FADH₂)-dependent enzyme that catalyzes the removal of a halogen atom from an unsaturated aliphatic organohalogen compound by the addition of a water molecule to the substrate. A soil bacterium, Pseudomonas sp. strain YL, inducibly produced a protein named Caa67YL when the cells were grown on 2-chloroacrylate (2-CAA). The caa67YL gene encoded a protein of 547 amino acid residues (Mr of 59,301), which shared weak but significant sequence similarity with various flavoenzymes and contained a nucleotide-binding motif. We found that 2-CAA is converted into pyruvate when the reaction was carried out with purified Caa67YL in the presence of FAD and a reducing agent [NAD(P)H or sodium dithionite] under anaerobic conditions. The reducing agent was not stoichiometrically consumed during this reaction, suggesting that FADH₂ is conserved by regeneration in the catalytic cycle. When the reaction was carried out in the presence of H₂¹⁸O, [¹⁸O]pyruvate was produced. These results indicate that Caa67YL catalyzes the hydration of 2-CAA to form 2-chloro-2-hydroxypropionate, which is chemically unstable and probably spontaneously dechlorinated to form pyruvate. 2-Bromoacrylate, but not other 2-CAA analogs such as acrylate and methacrylate, served as the substrate of Caa67YL. Thus, we named this new enzyme 2-haloacrylate hydratase. The enzyme is very unusual in that it requires the reduced form of FAD for hydration, which involves no net change in the redox state of the coenzyme or substrate.
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