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• W2054808510 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 (Mr 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. 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 (Mr 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. Organohalogen compounds are useful as herbicides, insecticides, plastics, solvents, and synthetic precursors and have been produced in large quantities by the chemical industry. In addition to these man-made compounds, >3,800 kinds of organohalogen compounds are produced biologically or by natural abiogenic processes, such as the eruption of volcanoes (1Gribble G.W. Chemosphere. 2003; 52: 289-297Crossref PubMed Scopus (454) Google Scholar). Although some of these compounds are persistent, others are biologically degraded in many ways to disappear from nature. To decompose these compounds, organisms, particularly microorganisms, have developed a variety of enzymes that act on organohalogen compounds (2Janssen D.B. Oppentocht J.E. Poelarends G.J. Curr. Opin. Biotechnol. 2001; 12: 254-258Crossref PubMed Scopus (161) Google Scholar, 3de Jong R.M. Dijkstra B.W. Curr. Opin. Struct. Biol. 2003; 13: 722-730Crossref PubMed Scopus (78) Google Scholar). Extensive studies have been carried out on these enzymes, partly because they are useful in the bioremediation of environments contaminated with organohalogen compounds (4Copley S.D. Curr. Opin. Chem. Biol. 1998; 2: 613-617Crossref PubMed Scopus (62) Google Scholar). These enzymes are also useful in the chemical industry in which organohalogen compounds are converted into other compounds in a regiospecific and stereospecific manner (5Swanson P.E. Curr. Opin. Biotechnol. 1999; 10: 365-369Crossref PubMed Scopus (99) Google Scholar). Various metabolic pathways for saturated organohalogen compounds and aromatic organohalogen compounds have been clarified (2Janssen D.B. Oppentocht J.E. Poelarends G.J. Curr. Opin. Biotechnol. 2001; 12: 254-258Crossref PubMed Scopus (161) Google Scholar, 3de Jong R.M. Dijkstra B.W. Curr. Opin. Struct. Biol. 2003; 13: 722-730Crossref PubMed Scopus (78) Google Scholar). For example, haloalkanes, 2-haloalkanoic acids, and haloalcohols are dehalogenated by haloalkane dehalogenase, 2-haloacid dehalogenase, and haloalcohol dehalogenase, respectively (6Janssen D.B. Curr. Opin. Chem. Biol. 2004; 8: 150-159Crossref PubMed Scopus (149) Google Scholar, 7Liu J.Q. Kurihara T. Miyagi M. Esaki N. Soda K. J. Biol. Chem. 1995; 270: 18309-18312Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 8Li Y.F. Hata Y. Fujii T. Hisano T. Nishihara M. Kurihara T. Esaki N. J. Biol. Chem. 1998; 273: 15035-15044Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 9Nardi-Dei V. Kurihara T. Park C. Miyagi M. Tsunasawa S. Soda K. Esaki N. J. Biol. Chem. 1999; 274: 20977-20981Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 10de Jong R.M. Tiesinga J.J. Rozeboom H.J. Kalk K.H. Tang L. Janssen D.B. Dijkstra B.W. EMBO J. 2003; 22: 4933-4944Crossref PubMed Scopus (101) Google Scholar). Fluoroacetate is hydrolyzed by fluoroacetate dehalogenase (11Liu J.Q. Kurihara T. Ichiyama S. Miyagi M. Tsunasawa S. Kawasaki H. Soda K. Esaki N. J. Biol. Chem. 1998; 273: 30897-30902Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). 4-Chlorobenzoate is converted into 4-chlorobenzoyl-CoA and then dehalogenated by 4-chlorobenzoyl-CoA dehalogenase (12Xu D. Wei Y. Wu J. Dunaway-Mariano D. Guo H. Cui Q. Gao J. J. Am. Chem. Soc. 2004; 126: 13649-13658Crossref PubMed Scopus (37) Google Scholar). Polychlorinated biphenyl is degraded by a series of enzymes, including oxygenases, dehydrogenases, and hydrolases (13Furukawa K. J. Gen. Appl. Microbiol. 2000; 46: 283-296Crossref PubMed Scopus (86) Google Scholar). Compared with the available information on the metabolisms of these organohalogen compounds, the information on the metabolisms of unsaturated aliphatic organohalogen compounds is limited. 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 (14Banerjee R. Ragsdale S.W. Annu. Rev. Biochem. 2003; 72: 209-247Crossref PubMed Scopus (593) Google Scholar), 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 (15de Jong R.M. Brugman W. Poelarends G.J. Whitman C.P. Dijkstra B.W. J. Biol. Chem. 2004; 279: 11546-11552Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 16Poelarends G.J. Serrano H. Person M.D. Johnson Jr., W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar, 17Poelarends G.J. Whitman C.P. Bioorg. Chem. 2004; 32: 376-392Crossref PubMed Scopus (43) Google Scholar). To gain insight into the biological systems that decompose unsaturated organohalogen compounds, we previously isolated bacterial strains that assimilate 2-chloroacrylate as a sole carbon source (18Hasan A. Takada H. Koshikawa H. Liu J. Kurihara T. Esaki N. Soda K. Biosci. Biotech. Biochem. 1994; 58: 1599-1602Crossref Scopus (28) Google Scholar). 2-Chloroacrylate is a bacterial metabolite of 2-chloroallyl alcohol, which is an intermediate or byproduct in industrial herbicide synthesis (19van der Waarde J.J. Kok R. Janssen D.B. Appl. Environ. Microbiol. 1993; 59: 528-535Crossref PubMed Google Scholar). Rats excrete 2-chloroacrylate when they are treated orally with herbicides containing a haloallyl substituent (20Marsden P.J. Casida J.E. J. Agric. Food Chem. 1982; 30: 627-631Crossref PubMed Scopus (38) Google Scholar). In addition, various halogenated acrylic acids are produced by red algae (21Woodlard F.X. Moore R.E. Roller P.P. Phytochemistry. 1979; 18: 617-620Crossref Scopus (52) Google Scholar). However, no enzyme catalyzing the conversion of 2-chloroacrylate has been identified. 2-Chloroacrylate structurally resembles 2-haloalkanoic acids but does not serve as the substrate for 2-haloacid dehalogenase (22Liu J.Q. Kurihara T. Hasan A.K. Nardi-Dei V. Koshikawa H. Esaki N. Soda K. Appl. Environ. Microbiol. 1994; 60: 2389-2393Crossref PubMed Google Scholar). Thus, a novel enzyme involved in the degradation of unsaturated organohalogen compounds was expected to be discovered from the studies on the metabolic pathway of 2-chloroacrylate. Here, we first identified the enzyme involved in the degradation of 2-chloroacrylate in a 2-chloroacrylate-grown bacterium, Burkholderia sp. WS (formerly Pseudomonas sp. WS) (18Hasan A. Takada H. Koshikawa H. Liu J. Kurihara T. Esaki N. Soda K. Biosci. Biotech. Biochem. 1994; 58: 1599-1602Crossref Scopus (28) Google Scholar, 23Kurata A. Kurihara T. Kamachi H. Esaki N. Tetrahedron: Asymmetry. 2004; 15: 2837-2839Crossref Scopus (24) Google Scholar). The enzyme catalyzed the reduction of 2-chloroacrylate to produce (S)-2-chloropropionate, which is supposed to be dehalogenated by (S)-2-haloacid dehalogenase. The enzyme is a new member of the medium-chain dehydrogenase/reductase superfamily with the highest sequence similarity with NADPH-dependent quinone oxidoreductase from Escherichia coli (24Riveros-Rosas H. Julian-Sanchez A. Villalobos-Molina R. Pardo J.P. Pina E. Eur. J. Biochem. 2003; 270: 3309-3334Crossref PubMed Scopus (88) Google Scholar, 25Thorn J.M. Barton J.D. Dixon N.E. Ollis D.L. Edwards K.J. J. Mol. Biol. 1995; 249: 785-799Crossref PubMed Scopus (97) Google Scholar). The enzyme specifically acts on organohalogen compounds, and we named the enzyme 2-haloacrylate reductase. This study revealed a new pathway for the biodegradation of unsaturated organohalogen compounds. Moreover, the enzyme is useful for the production of (S)-2-chloropropionate, which is used in a large quantity as a synthetic precursor for the industrial production of aryloxyphenoxypropionic acid herbicides (26Haga T. Crosby K.E. Schussler J.R. Palmer C.J. Yoshii H. Kimura F. Kurihara N. Miyamoto J. Chirality in Agrochemicals. John Wiley & Sons Ltd., Chichester, West Sussex, United Kingdom1998: 175-197Google Scholar). Materials—2-Chloroacrylic acid was purchased from Lancaster Synthesis Ltd. (Lancashire, United Kingdom). (S)-2-Chloropropionic acid and (R)-2-chloropropionic acid were from Nacalai Tesque (Kyoto, Japan). All of the other chemicals were of analytical grade. Microorganism and Culture Conditions—Burkholderia sp. WS was grown aerobically at 28 °C in a medium containing 0.2% (NH4)2SO4, 0.05% Bacto yeast extract (Difco), 0.1% K2HPO4, 0.1% NaH2PO4, and 0.01% MgSO4·7H2O (pH 7.1). Either 0.2% sodium 2-chloroacrylate or 0.2% sodium lactate was added to the medium as a carbon source. Preparation of the Crude Extract of Burkholderia sp. WS—Cells of Burkholderia sp. WS were harvested at the late logarithmic phase, washed with a standard buffer (a 60 mm potassium phosphate buffer containing 1 mm dithiothreitol, pH 7.1), resuspended in the standard buffer, and disrupted by sonication. Cell debris was removed by centrifugation (18,000 × g for 10 min at 4 °C), and the crude extract was stored at –80 °C. Two-dimensional Polyacrylamide Gel Electrophoresis—Proteins of 2-chloroacrylate- and lactate-grown cells were compared by two-dimensional PAGE. Isoelectric focusing (IEF) 1The abbreviations used are: IEF, isoelectric focusing; MS, mass spectrometry. was performed in a vertical disc format as follows. The lower reservoir was filled with 10 mm H3PO4, and the upper reservoir was filled with 20 mm NaOH. The IEF gel mixture contained 8 m urea, 2% Nonidet P-40, and 2.0% Bio-Lyte, pH 3–10 (Bio-Rad) in 0.8% polyacrylamide gel with N,N′-methylene-bisacrylamide as a cross linker. The gel was then consecutively pre-run at 200 V for 15 min, 300 V for 15 min, and 400 V for 30 min. After pre-run, the crude cell extract dissolved in 8 m urea, 1% Nonidet P-40, 2% Bio-Lyte, pH 3–10, and 0.05% 2-mercaptoethanol was loaded onto the IEF gel. The IEF gel was run at 400 V for 12 h and then at 800 V for 1 h. After isoelectric focusing, the gel was treated with an equilibration buffer (62.5 mm Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate, and 0.001% bromphenol blue) for 20 min. The equilibrated IEF gel was subjected to SDS-PAGE. The gel was stained with Coomassie Brilliant Blue R-250. Amino Acid Sequencing—To determine the amino acid sequences of the 2-chloroacrylate-inducible proteins (CAA43 and CAA67), proteins of the 2-chloroacrylate-grown cells were separated by two-dimensional PAGE. For N-terminal amino acid sequencing, the proteins in the gel were blotted onto an Immobilon-P membrane (Millipore, Bedford, MA) and stained with Coomassie Brilliant Blue R-250. The spots were excised, and the N-terminal amino acid sequences were determined with a Shimadzu PPSQ-21 protein sequencer (Kyoto, Japan). Internal amino acid sequencing was performed by the APRO Life Science Institute, Inc. (Naruto, Japan). Gene Cloning—A part of the CAA43 gene was amplified by degenerate PCR with a sense primer (5′-ATGGCIGCIGTIATHCAYAA-3′), an antisense primer (5′-CCIGCIARRTGIACRTCGTC-3′), the genomic DNA of Burkholderia sp. WS, and TaKaRa LA TaqDNA polymerase (Takara Bio, Otsu, Japan). The program for the PCR was as follows: denaturation at 95 °C for 3 min followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 47.9 °C for 1 min, and extension at 72 °C for 2 min. The 3′-region of the CAA43 gene was obtained by using an LA PCR in vitro cloning kit (Takara Shuzo). XbaI was used for the digestion of the genomic DNA. The oligonucleotides 5′-CGCCCCTCGGTGCCTACAGC-3′ and 5′-CTACCCCGCCGAAAAACTGA-3′, which anneal to the internal region of the CAA43 gene, were used for the first and the second PCR, respectively. The 5′-region of the CAA43 gene was obtained by inverse PCR. PCR was performed using primers 5′-CCGGGCGAGCCAACCTTAAC-3′ and 5′-CGCCCCTCGGTGCCTACAGC-3′ and the self-ligated SalI-digested genomic DNA as a template with the following program: denaturation at 94 °C for 4 min followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 4 min. The amplified fragment contained the 3′-region of the CAA67 gene. The 5′-region of the CAA67 gene was obtained by using an LA PCR in vitro cloning kit with PstI-digested genomic DNA. Primers 5′-CATGAAATTCGGGACACACT-3′ and 5′-AGAAGGTGCCTGTGAGTGAA-3′ were used for the first and the second PCR, respectively. Expression of CAA43 and CAA67 with Recombinant E. coli Cells—A DNA fragment coding for CAA43 and CAA67 was amplified by PCR with primers 5′-CACCAGGAGGAGGATGTTCATGTCGG-3′ and 5′-CTACGCTTGCGGAAGCAAA-3′ and the genomic DNA of Burkholderia sp. WS as a template. The program was as follows: denaturation at 94 °C for 120 s followed by 30 cycles of 94 °C for 15 s, 61.1 °C for 30 s, and 68 °C for 210 s. A DNA fragment coding for CAA43 was amplified by PCR with primers 5′-CACCAGGAGACTTTATCATGGTAATGG-3′ and 5′-CTACGCTTGCGGAAGCAAA-3′ and the genomic DNA as a template. The program was as follows: denaturation at 94 °C for 120 s followed by 30 cycles of 94 °C for 15 s, 57.5 °C for 30 s, and 68 °C for 80 s. The amplified products were cloned into the expression vector pET101/D-TOPO (Invitrogen) downstream of the T7 promoter. The plasmid for the expression of the CAA43 gene was named pET101/D-TOPO-CAA43, and that for the co-expression of the CAA43 gene and the CAA67 gene was named pET101/D-TOPO-CAA67&CAA43. These plasmids were introduced into E. coli BL21(DE3). Each of the recombinants was grown in an LB medium containing 50 μg/ml ampicillin at 37 °C. When the absorbance at 600 nm reached 0.5, isopropyl-1-thio-β-d-galactopyranoside was added to the culture medium to a final concentration of 1 mm. After cultivation for 4 h at 37 °C in the presence of isopropyl-1-thio-β-d-galactopyranoside, the cells were harvested, suspended in the standard buffer, and disrupted by sonication. The cellular debris was removed by centrifugation. Purification of CAA43—Recombinant E. coli BL21(DE3) cells harboring pET101/D-TOPO-CAA67&CAA43 were grown in a 1-liter LB medium containing 50 μg/ml ampicillin at 37 °C. When the absorbance at 600 nm reached 0.6, isopropyl-1-thio-β-d-galactopyranoside was added to the medium to a final concentration of 1 mm. After additional cultivation for 32 h, the cells were harvested, washed with the standard buffer, and stored at –80 °C until use. The stored cells were suspended with 80 ml of the standard buffer and disrupted by sonication. The cellular debris was removed by centrifugation, and the supernatant was used immediately for the purification of CAA43. All of the purification procedures were carried out at 4 °C. The cell-free extract was applied to a TOYOPEARL DEAE-650M column (2.5 × 15 cm) (Tosoh, Tokyo, Japan) equilibrated with the standard buffer. CAA43 was eluted with 400 ml of the standard buffer, and the active fractions were collected. The enzyme fraction was applied to an Orange-Sepharose CL-4B column (3 × 10 cm) equilibrated with the standard buffer. After the column was washed with 160 ml of the same buffer, the enzyme was eluted with a linear gradient of a 60–500 mm potassium phosphate buffer, pH 7.1, containing 1 mm dithiothreitol with a total volume of 400 ml. The purified enzyme was dialyzed against the standard buffer and stored at –80 °C. Enzyme Assay—The standard assay mixture for 2-haloacrylate reductase contained a 60 mm Tris sulfate buffer, pH 8.0, 5 mm 2-chloroacrylate, 0.6 mm NADPH, and enzyme. Absorbance at 340 nm was monitored at 35 °C. One unit of the enzyme activity was defined as the amount of enzyme that catalyzes the oxidation of 1 μmol of NADPH/min. To determine the enzyme activity toward other substrates, 2-chloroacrylate was replaced by the respective substrates of the same concentration. Ethanol was added to a final concentration of 1% for water-insoluble substrates. The decrease of 2-chloroacrylate and the increase of (S)-2-chloropropionate in the course of the enzyme reaction were monitored by electrospray ionization mass spectrometry as follows. A 500-μl reaction mixture containing a 60 mm ammonium acetate buffer, pH 7.1, 5 mm 2-chloroacrylate, 5 mm NADPH, and enzyme was incubated at 30 °C. The reaction was terminated by the addition of 1 ml of acetonitrile. The reaction mixture was centrifuged, filtrated, diluted with acetonitrile/10 mm ammonium acetate (1:1), and then introduced into a mass spectrometer (API3000 liquid chromatography-mass spectrometry (MS)/MS system, PE Sciex, Foster City, CA) at 5 μl/min. The molecular ions of 2-[35Cl]chloroacrylate and 2-[37Cl]chloropropionate were detected in the negative ion mode at m/z 105 and 109, respectively. Determination of the Configuration of 2-Chloropropionate Enzymatically Produced—A 500-μl reaction mixture containing a 60 mm ammonium acetate buffer, pH 7.1, 5 mm 2-chloroacrylate, 5 mm NADPH, and enzyme was incubated at 30 °C. After overnight incubation, the pH of the reaction mixture was adjusted to 1 with 200 μl of 6 n HCl. 2-Chloropropionic acid in the mixture was extracted with 700 μl of ethyl acetate and converted into methyl ester with trimethylsilyldiazomethane. The configuration of the methyl 2-chloropropionate was determined with a gas chromatography-MS system (AutoSystem XL Gas Chromatograph, TurboMass Mass Spectrometer, PerkinElmer Life Sciences) equipped with a WCOT-fused silica column (0.32 mm × 25 m, Varian, Palo Alto, CA). The injection port was maintained at 200 °C. The column temperature program was 40 °C for 2 min, 5 °C/min to 70 °C, and 70 °C for 4 min. The ion source was maintained at 220 °C. Helium was used as a carrier gas at 0.63 p.s.i. The mass spectrometer was operated with an ionization voltage of 70 eV. In the selective ion-monitoring mode, m/z of 59, 63, 87, 91, and 122 were used to detect 2-chloropropionate methyl ester. Methyl-(S)-2-chloropropionate and methyl (R)-2-chloropropionate were eluted at 5.5 and 6.0 min, respectively. Determination of the Kinetic Parameters—To determine the apparent Vmax and Km values of the enzyme, the standard assay was performed with various concentrations of substrates as follows: 0.1–10 mm sodium 2-chloroacrylate; 0.1–10 mm sodium 2-bromoacrylate; and 0.05–0.5 mm NADPH. Effects of pH and Temperature on the Enzyme Activity—To examine the effect of pH on the enzyme stability, the standard assay was performed with the enzyme preincubated for 30 min at 30 °C in the following buffers (60 mm): citrate-NaOH (pH 5.5–6.5); potassium phosphate (pH 6.0–8.0); Tris sulfate (pH 7.0–9.0); and glycine-NaOH (pH 9.0–10.5). To examine the effect of pH on the enzyme activity, the initial reaction velocities were measured by the standard assay method with the buffers (60 mm) of various pH values listed above. The effect of the temperature on the stability of the enzyme was determined by incubating the enzyme at different temperatures from 25 to 50 °C for 30 min prior to the standard assay. The effect of the temperature on the enzyme activity was determined by performing the standard assay at different temperatures from 25 to 50 °C. Molecular Weight Determination—The subunit molecular weight of the enzyme was analyzed by SDS-PAGE and by mass spectrometry with an API3000 liquid chromatography-MS/MS system. The molecular weight of the native enzyme was analyzed by gel filtration with an ÄKTA Explorer system (Amersham Biosciences) equipped with a Superose 12 10/300 GL column (Amersham Biosciences). Protein Assay—The protein concentration was determined with a protein assay kit (Nacalai Tesque) according to the manufacturer's instructions with bovine serum albumin as a standard. NADPH-dependent Reduction of 2-Chloroacrylate and Inducible Nature of the Enzyme Catalyzing This Reaction—We found that 2-chloroacrylate is converted into 2-chloropropionate in the presence of NADPH and the cell-free extract of 2-chloroacrylate-grown Burkholderia sp. WS (Fig. 1, A–C). We also confirmed that NADPH is converted into NADP+ under the same condition (data not shown). The results suggest that an NADPH-dependent reductase acting on 2-chloroacrylate occurs in the 2-chloroacrylate-grown cells. Other reducing agents, such as NADH and the reduced form of FAD, did not substitute for NADPH (data not shown). The increase of peaks with m/z values of 89 and 87 (Fig. 1, B and C) suggests that 2-chloropropionate is further metabolized to lactate (m/z = 89) and pyruvate (m/z = 87). We next examined whether the enzyme is constitutively produced or inducibly produced when the cells are grown on 2-chloroacrylate. As shown in Fig. 1D, 2-chloroacrylate was not degraded when the cell-free extract from the lactate-grown cells was used. 2-Chloroacrylate-dependent oxidation of NADPH was not observed under this condition. Thus, the enzyme catalyzing the reduction of 2-chloroacrylate to produce 2-chloropropionate is inducibly synthesized when the cells are grown on 2-chloroacrylate as a carbon source. Identification of Proteins Inducibly Synthesized by 2-Chloroacrylate—Proteins from 2-chloroacrylate-grown cells and lactate-grown cells were compared by two-dimensional PAGE to identify proteins inducibly synthesized in Burkholderia sp. WS grown on 2-chloroacrylate. Two major proteins (Fig. 2, arrowheads) were found only in the 2-chloroacrylate-grown cells, suggesting their involvement in the metabolism of 2-chloroacrylate. These proteins were named CAA43 and CAA67, as indicated in Fig. 2. The N-terminal amino acid sequences of CAA43 and CAA67 were VMAAVIHKKGGPDNFV and XDVLVTDVLVVXE, respectively, and the following internal amino acid sequences were determined: DLDLDDVHLAGLMLK (CAA43) and VFVDFRETKPEEWAPDSLTGTFLGK (CAA67). Cloning of the Genes Coding for CAA43 and CAA67—Degenerate primers were designed based on the amino acid sequences of CAA43 and CAA67, and the genes coding for these proteins were cloned as described under “Experimental Procedures.” The genes coding for CAA43 and CAA67 were found to be located next to each other on the genome of Burkholderia sp. WS (GenBank™ accession number AB196962). The CAA43 gene was located downstream of the CAA67 gene, and their distance was 267 bp. The CAA43 gene contained an open reading frame of 1,002 nucleotides coding for 333 amino acid residues (Mr 35,788), and the CAA67 gene contained an open reading frame of 1,644 nucleotides coding for 547 amino acid residues (Mr 58,684). Putative Shine-Dalgarno sequences, AGGAGG and AGGAG, were found in the upstream regions of the initiation codons of the CAA67 gene and the CAA43 gene, respectively. Possible –35 (CTTGATGT) and –10 (TTTAAT) sequences were found only in the upstream region of the CAA67 gene. Structural Characteristics of CAA43 and CAA67—CAA43 shares the highest sequence similarity with NADPH-dependent quinone oxidoreductase from E. coli (NCBI accession number 131763) (38.2% identity), which catalyzes the NADPH-dependent reduction of various quinones, among the proteins in the data base with known function (Fig. 3) (25Thorn J.M. Barton J.D. Dixon N.E. Ollis D.L. Edwards K.J. J. Mol. Biol. 1995; 249: 785-799Crossref PubMed Scopus (97) Google Scholar, 27Edwards K.J. Barton J.D. Rossjohn J. Thorn J.M. Taylor G.L. Ollis D.L. Arch. Biochem. Biophys. 1996; 328: 173-183Crossref PubMed Scopus (63) Google Scholar). CAA43 has a nucleotide binding motif, AXXGXXG, in the region from 153 to 159 and is supposed to bind to NADH or NADPH. The crystal structure of the quinone oxidoreductase from E. coli complexed with NADPH showed that the 2′-phosphate group of NADPH is surrounded by polar and positively charged residues, Lys177, Tyr192, and Arg317 (25Thorn J.M. Barton J.D. Dixon N.E. Ollis D.L. Edwards K.J. J. Mol. Biol. 1995; 249: 785-799Crossref PubMed Scopus (97) Google Scholar). These residues provide a positively charged pocket that accepts the phosphate group. All of these residues are conserved in CAA43, suggesting that CAA43 is an NADPH-dependent enzyme. CAA67 has a weak but significant sequence similarity with l-aspartate oxidase from E. coli (NCBI accession number 5542180) (17.6% identity) and fumarate reductase (subunit A) from Wolinella succinogenes (NCBI accession number 37538290) (17.2% identity), which require FAD as a cofactor (data not shown) (28Bossi R.T. Negri A. Tedeschi G. Mattevi A. Biochemistry. 2002; 41: 3018-3024Crossref PubMed Scopus (59) Google Scholar, 29Lancaster C.R. Kroger A. Auer M. Michel H. Nature. 1999; 402: 377-385Crossref PubMed Scopus (311) Google Scholar). Reduction of 2-Chloroacrylate Catalyzed by CAA43—The amino acid sequence analyses suggested that CAA43 and CAA67 require NADPH and FAD, respectively, as a cofactor. Thus, CAA43 was supposed to be involved in the reduction of 2-chloroacrylate because NADPH was required for the reduction of 2-chloroacrylate by the extract of the 2-chloroacrylate-grown cells as described above. We constructed a recombinant E. coli strain that produces CAA43 under the control of the T7 promoter to see whether CAA43 catalyzes the reduction of 2-chloroacrylate. We measured the 2-chloroacrylate-reducing activity in the cell-free extracts prepared from the recombinant E. coli cells producing CAA43 and the cells not producing CAA43 by electrospray ionization mass spectrometry (data not shown). When the extract containing CAA43 was used, the peak corresponding to 2-[35Cl]chloroacrylate (m/z = 105) was decreased and the peak corresponding to 2-[37Cl]chloropropionate (m/z = 109) was increased. The reaction did not proceed in the absence of CAA43. The result indicates that CAA43 catalyzes the conversion of 2-chloroacrylate to 2-chloropropionate. Purification of CAA43—E. coli BL21 (DE3) harboring pET101/D-TOPO-CAA67&CAA43 was used for purification of CAA43, because the amount of CAA43 was more abundant in the cells harboring pET101/D-TOPO-CAA67&CAA43 than in the cells harboring pET101/D-TOPO-CAA43 (data not shown). Although the cells harboring pET101/D-TOPO-CAA67&CAA43 produced both CAA43 and CAA67, the majority of CAA67 was recovered in the insoluble fraction when the cell-free extract was prepared. Table I summarizes the results of the purification of CAA43. The enzyme was purified 5.0-fold with 30% recovery, and the purified enzyme was found to be homogeneous by SDS-PAGE (data not shown). CAA67 was not co-purified with CAA43.Table IPurification of CAA43 from recombinant E. coli BL21(DE3) cells harboring pET101/D-TOPO-CAA67&CAA43Purification stepTotal activityTotal proteinSpecific activityYieldPurificationunitsmgunits/mg%-foldCrude extract33830.401001TOYOPEARL DEAE22171.3673.3Orange-Sepharose105.02.0305.0 Open table in a new tab The purified CAA43 was shown to catalyze the conversion of 2-chloroacrylate into 2-chloropropionate by electrospray mass spectrometric analysis (Fig. 4). 1 mol of NADPH was converted into NADP+ when 1 mol of 2-chloroacrylate was converted into 2-chloropropionate. Molecular Weight and Subunit Structure of CAA43—The purified CAA43 lacked the N-terminal methionine residue. The molecular weight of the subunit of CAA43 was estimated to be ∼37,000 by SDS-PAGE, which is in good agreement with the value (35,656) calculated from the primary structure of CAA43 without the N-terminal methionine residue. Mass spectrometric analysis indicated that the molecular weight of the purified CAA43 was 35,695, suggesting the association of a potassium ion to the enzyme. Gel filtration analysis showed that the molecular weight of the native enzyme is ∼30,000, suggesting that the enzyme is monomeric. Effects of pH and Temperature on CAA43—The effect of pH on the activity of CAA43 was examined over the pH range from 5.5 to 10.5, and an optimum pH was found to be ∼8.0. CAA43 was stable between pH 7.0 and 8.0 for 30 min at 30 °C. CAA43 exhibited its maximum activity at 40 °C and was stable at 35 °C or lower temperatures for 30 min. Determination of the Configuration of 2-Chloropropionate Produced with CAA43—2-Chloroacrylate was incubated with the purified CAA43 in the presence of NADPH. After the reaction, the product was esterified and analyzed by gas chromatography-MS with a chiral resolution column. The product was co-eluted with (S)-2-chloropropionate methyl ester. A molecular ion of m/z 122 and fragment ions of m/z 59, 63, 87, and 91, which are identical to those of authentic (S)-2-chloropropionate methyl ester, were found in the mass spectrum of the product (data not shown). (R)-2-Chloropropionate methyl ester was not detected in the chromatogram of the product. Thus, CAA43 catalyzes the asymmetric reduction of 2-chloroacrylate to produce (S)-2-chloropropionate. Substrate and Cofactor Specificity—The substrate specificity of CAA43 was examined. The enzyme acted on 2-chloroacrylate and 2-bromoacrylate, but 2-fluoroacrylate did not serve as the substrate. The specific activity toward 2-bromoacrylate was comparable to that toward 2-chloroacrylate. The apparent Km and Vmax values for 2-chloroacrylate were 0.45 mm and 1.9 μmol/min/mg, respectively. The apparent Km and Vmax values for 2-bromoacrylate were 0.19 mm and 2.8 μmol/min/mg, respectively. Although the velocity versus substrate plot for 2-chloroacrylate showed typical Michaelis-Menten kinetics, the reductase activity was inhibited by the high concentration (>2.5 mm) of 2-bromoacrylate. Analogs of 2-chloroacrylate, such as acrylate, methacrylate, 2-cloro-1-propene, 2-chloroacrylonitrile, phosphoenolpyruvate, and fumarate, were inert as substrates, suggesting that a free carboxyl group and a halogen atom bound to the α-position of acrylic acid are required for the substrates. Although CAA43 resembles NADPH-dependent quinone oxidoreductase in its primary structure, CAA43 did not act on 1,4-benzoquinone and 1,4-naphthoquinone, which serve as the substrates of the quinone oxidoreductase (25Thorn J.M. Barton J.D. Dixon N.E. Ollis D.L. Edwards K.J. J. Mol. Biol. 1995; 249: 785-799Crossref PubMed Scopus (97) Google Scholar, 30Shimomura Y. Sumiguchi-Agari K. Masui R. Kuramitsu S. Fukuyama K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1365-1367Crossref PubMed Scopus (4) Google Scholar, 31Rao P.V. Krishna C.M. Zigler Jr., J.S. J. Biol. Chem. 1992; 267: 96-102Abstract Full Text PDF PubMed Google Scholar). As expected from the primary structure, CAA43 required NADPH for its catalytic activity and no reductase activity was observed when NADH was used instead of NADPH. The apparent Km value for NADPH was 0.090 mm. Occurrence of 2-Haloacrylate Reductase—We discovered an NADPH-dependent enzyme that catalyzes the reduction of a carbon-carbon double bond of 2-chloroacrylate and 2-bromoacrylate. 2-Haloacrylate analogs such as acrylate and methacrylate did not serve as the substrate. The inducible nature of the enzyme in Burkholderia sp. WS suggests that 2-haloacrylate is a physiological substrate of this enzyme. Although the enzyme shares significant sequence similarity with NADPH-dependent quinone oxidoreductase from E. coli (25Thorn J.M. Barton J.D. Dixon N.E. Ollis D.L. Edwards K.J. J. Mol. Biol. 1995; 249: 785-799Crossref PubMed Scopus (97) Google Scholar), the enzyme is clearly different from quinone oxidoreductase in its substrate specificity. We named this novel enzyme 2-haloacrylate reductase. NADPH-dependent quinone oxidoreductases belong to the medium-chain dehydrogenase/reductase superfamily (24Riveros-Rosas H. Julian-Sanchez A. Villalobos-Molina R. Pardo J.P. Pina E. Eur. J. Biochem. 2003; 270: 3309-3334Crossref PubMed Scopus (88) Google Scholar), which includes proteins such as ζ-crystallin (31Rao P.V. Krishna C.M. Zigler Jr., J.S. J. Biol. Chem. 1992; 267: 96-102Abstract Full Text PDF PubMed Google Scholar) and zinc-dependent alcohol dehydrogenase (32LeBrun L.A. Park D.H. Ramaswamy S. Plapp B.V. Biochemistry. 2004; 43: 3014-3026Crossref PubMed Scopus (48) Google Scholar). Among these proteins, quinone oxidoreductase from E. coli shares the highest sequence similarity with 2-haloacrylate reductase (38.2%) (25Thorn J.M. Barton J.D. Dixon N.E. Ollis D.L. Edwards K.J. J. Mol. Biol. 1995; 249: 785-799Crossref PubMed Scopus (97) Google Scholar). Other proteins in the superfamily are much more distantly related to 2-haloacrylate reductase. For example, ζ-crystallin from guinea pig (NCBI accession number 117549) and alcohol dehydrogenase from horse liver (NCBI accession number 625197) share only 22.5 and 15.6% sequence identities, respectively, with 2-haloacrylate reductase (31Rao P.V. Krishna C.M. Zigler Jr., J.S. J. Biol. Chem. 1992; 267: 96-102Abstract Full Text PDF PubMed Google Scholar, 32LeBrun L.A. Park D.H. Ramaswamy S. Plapp B.V. Biochemistry. 2004; 43: 3014-3026Crossref PubMed Scopus (48) Google Scholar). Metabolism of 2-Haloacrylate—We detected the activity of (S)-2-haloacid dehalogenase, which catalyzes the hydrolytic dehalogenation of (S)-2-haloalkanoic acids (7Liu J.Q. Kurihara T. Miyagi M. Esaki N. Soda K. J. Biol. Chem. 1995; 270: 18309-18312Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 8Li Y.F. Hata Y. Fujii T. Hisano T. Nishihara M. Kurihara T. Esaki N. J. Biol. Chem. 1998; 273: 15035-15044Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), in the crude extract of 2-chloroacrylate-grown Burkholderia sp. WS (data not shown), although we previously failed to detect the enzyme because of its low activity (18Hasan A. Takada H. Koshikawa H. Liu J. Kurihara T. Esaki N. Soda K. Biosci. Biotech. Biochem. 1994; 58: 1599-1602Crossref Scopus (28) Google Scholar). We also identified the gene coding for this enzyme (GenBank™ accession number AB196963). Recombinant E. coli cells expressing this gene exhibited dehalogenase activity. Thus, in Burkholderia sp. WS, 2-haloacrylate reductase probably catalyzes the first step of the assimilation of 2-chloroacrylate to produce (S)-2-chloropropionate, which is subsequently hydrolyzed to (R)-lactate by (S)-2-haloacid dehalogenase (Fig. 5). Janssen and co-workers (19van der Waarde J.J. Kok R. Janssen D.B. Appl. Environ. Microbiol. 1993; 59: 528-535Crossref PubMed Google Scholar) isolated a 2-chloroallylalcohol-assimilating bacterium, Pseudomonas sp. JD2, in which 2-chloroallylalcohol is proposed to be degraded via 2-chloroacrylate. Although the metabolic pathway for 2-chloroacrylate and the enzymes involved in this pathway are unidentified, 2-haloacrylate reductase may occur in this bacterium and catalyze the reduction of 2-chloroacrylate to produce 2-chloropropionate. The presence of dehalogenase that acts on 2-chloropropionate in this bacterium supports this speculation. 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 (15de Jong R.M. Brugman W. Poelarends G.J. Whitman C.P. Dijkstra B.W. J. Biol. Chem. 2004; 279: 11546-11552Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 16Poelarends G.J. Serrano H. Person M.D. Johnson Jr., W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar, 17Poelarends G.J. Whitman C.P. Bioorg. Chem. 2004; 32: 376-392Crossref PubMed Scopus (43) Google Scholar). In this reaction, 3-haloacrylate is first hydrated to form an unstable halohydrin species, which subsequently degrades to produce malonate semialdehyde. Unlike this reaction, 2-haloacrylate is first reduced and then dehalogenated by two distinct enzymes in Burkholderia sp. WS. Possible Function of CAA67—The CAA67 gene is located in the immediate upstream region of the 2-haloacrylate reductase gene. Because the –35 and –10 sequences were found only in the upstream region of the CAA67 gene, the two genes probably constitute an operon. The fact that CAA67 is inducibly synthesized together with 2-haloacrylate reductase when the cells are grown on 2-chloroacrylate supports this view. Thus, CAA67 probably plays a role in the assimilation of 2-chloroacrylate. Because CAA67 shares sequence similarity with FAD-dependent enzymes such as fumarate reductase and l-aspartate oxidase (28Bossi R.T. Negri A. Tedeschi G. Mattevi A. Biochemistry. 2002; 41: 3018-3024Crossref PubMed Scopus (59) Google Scholar, 29Lancaster C.R. Kroger A. Auer M. Michel H. Nature. 1999; 402: 377-385Crossref PubMed Scopus (311) Google Scholar), it is reasonable to assume that CAA67 is an oxidoreductase functioning in the metabolic pathway of 2-chloroacrylate. Perhaps it is involved in the regeneration of NADPH consumed by 2-haloacrylate reductase or in the catabolism of (S)-2-chloropropionate produced by 2-haloacrylate reductase (Fig. 5). We currently have no experimental data that allow us to determine the role of CAA67. The function of CAA67 is now under investigation by biochemical analysis of the purified protein as well as by disruption of the CAA67 gene. Application of 2-Haloacrylate Reductase to the Production of Chiral Compounds—2-Chloropropionate is a building block for the synthesis of the aryloxyphenoxypropionic acid herbicide, which is one of the most abundantly used herbicides in the world (26Haga T. Crosby K.E. Schussler J.R. Palmer C.J. Yoshii H. Kimura F. Kurihara N. Miyamoto J. Chirality in Agrochemicals. John Wiley & Sons Ltd., Chichester, West Sussex, United Kingdom1998: 175-197Google Scholar). Because the (R)-isomer of the aryloxyphenoxypropionic acid has the herbicidal activity, it is desirable to use (S)-2-chloropropionate for the production of the herbicide (note that the reaction proceeds with the inversion of the configuration). (S)-2-Chloropropionate is currently produced by optical resolution in which the (R)-isomer of a racemic 2-chloropropionate is selectively degraded with (R)-2-haloacid dehalogenase (33Breuer M. Ditrich K. Habicher T. Hauer B. Kesseler M. Sturmer R. Zelinski T. Angew. Chem. Int. Ed. Engl. 2004; 43: 788-824Crossref PubMed Scopus (1175) Google Scholar). Because the theoretical maximum yield of (S)-2-chloropropionate by this procedure is only 50%, it is desirable to develop a new method to produce (S)-2-chloropropionate in a much better yield. 2-Haloacrylate reductase catalyzes the asymmetric reduction of 2-chloroacrylate to produce (S)-2-chloropropionate and may be used for this purpose. The system for the production of (S)-2-chloropropionate is under construction by coupling the 2-haloacrylate reductase reaction with an NADPH-regeneration system." @default.
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• W2054808510 cites W1987706462 @default.
• W2054808510 cites W2000063610 @default.
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• W2054808510 cites W2007890857 @default.
• W2054808510 cites W2008584836 @default.
• W2054808510 cites W2017334329 @default.
• W2054808510 cites W2018012868 @default.
• W2054808510 cites W2021385460 @default.
• W2054808510 cites W2027428191 @default.
• W2054808510 cites W2027916840 @default.
• W2054808510 cites W2034321085 @default.
• W2054808510 cites W2034550552 @default.
• W2054808510 cites W2039691050 @default.
• W2054808510 cites W2054618890 @default.
• W2054808510 cites W2060432205 @default.
• W2054808510 cites W2071124913 @default.
• W2054808510 cites W2086700751 @default.
• W2054808510 cites W2097444598 @default.
• W2054808510 cites W2104215735 @default.
• W2054808510 cites W2143351258 @default.
• W2054808510 cites W2151449293 @default.
• W2054808510 cites W2162245062 @default.
• W2054808510 cites W2168556731 @default.
• W2054808510 cites W2169573274 @default.
• W2054808510 cites W2169575504 @default.
• W2054808510 cites W4252114087 @default.
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