2-haloacrylate hydratase, a new class of flavoenzyme that catalyzes the addition of water to the substrate for dehalogenation

Abstract

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) (FADH(2))-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 Caa67(YL) when the cells were grown on 2-chloroacrylate (2-CAA). The caa67(YL) gene encoded a protein of 547 amino acid residues (M(r) 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 Caa67(YL) 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 FADH(2) is conserved by regeneration in the catalytic cycle. When the reaction was carried out in the presence of H(2)(18)O, [(18)O]pyruvate was produced. These results indicate that Caa67(YL) 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 Caa67(YL). 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.

FIG. 1.

Two-dimensional PAGE analysis of the proteins of 2-CAA- and lactate-grown Pseudomonas sp. YL. Soluble proteins from 2-CAA-grown cells (A) and lactate-grown cells (B) were analyzed. The arrowhead indicates the spot of a 2-CAA-inducible protein, which we named Caa67_YL.

FIG. 2.

SDS-PAGE analysis of Caa67_YL. Purified protein (10 μg) was loaded onto the gel.

FIG. 3.

Mass spectrometric monitoring of the conversion of 2-CAA with Caa67_YL. 2-CAA was incubated with or without purified Caa67_YL in the presence of 0.1 mM FAD and 10 mM NADH under anaerobic conditions as described in Materials and Methods. (A and B) The mixture was analyzed after a 4-h reaction without (A) and with (B) the addition of Caa67_YL by ESI-MS in the negative-ion mode. (C) To determine the incorporation of an oxygen atom of a water molecule into the substrate, the reaction was carried out in the presence of 50% H₂¹⁸O, and the solution was analyzed after a 4-h reaction. Because chlorine has two isotopes with mass numbers of 35 and 37 at a ratio of 3:1, 2-CAA has two peaks at m/z 105 and 107. The peaks at m/z 87 and 89 are due to pyruvate and ¹⁸O-labeled pyruvate, respectively, as described in the text. The peak at m/z 97 is due to hydrogen sulfate in the reaction buffer and dihydrogen phosphate in the enzyme preparation.

FIG. 4.

Effects of pH and temperature on Caa67_YL. (A and B) Effects of pH on the activity (A) and stability (B) were determined by using the following buffers (60 mM): citrate-NaOH (closed circles) (pH 5.5 to 6.5), potassium phosphate (closed squares) (pH 6.5 to 8.0), Tris-sulfate (closed triangles) (pH 8.0 to 9.0), and glycine-NaOH (closed diamonds) (pH 9.0 to 10.5). The enzyme activity after treatment with Tris-sulfate (pH 9.0) was taken as 100% in A. (C and D) Effects of temperature on the activity (C) and stability (D) were determined as described in Materials and Methods. The enzyme activity at 40°C was taken as 100% in C.

FIG. 5.

Amount of NADH consumed for dehalogenation of 2-CAA. 2-CAA was incubated with Caa67_YL in the presence of 0.1 mM FAD and 10 mM NADH under anaerobic conditions, and the consumption of NADH and the formation of chloride ions were monitored. The concentrations of NADH and chloride ions in the reaction mixture are indicated by closed triangles and closed circles, respectively.

FIG. 6.

Reaction catalyzed by 2-haloacrylate hydratase.