Mass spectrometric analysis of the reactions catalyzed by -2-haloacid dehalogenase mutants and implications for the roles of the catalytic amino acid residues

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of -2-haloalkanoic acids to produce the corresponding -2-hydroxyalkanoic acids. Asp10 of -2-haloacid dehalogenase from Pseudomonas sp. YL nucleophilically attacks the α-carbon atom of the substrate to form an ester intermediate, which is subsequently hydrolyzed by an activated water molecule. We previously showed that the replacement of Thr14, Arg41, Ser118, Lys151, Tyr157, Ser175, Asn177, and Asp180 causes significant loss in the enzyme activity, indicating the involvement of these residues in catalysis. In the present study, we tried to determine which process these residues are involved in by monitoring the formation of the ester intermediate by measuring the molecular masses of the mutant enzymes using ionspray mass spectrometry. When the wild-type enzyme and the T14A, S118D, K151R, Y157F, S175A, and N177D mutant enzymes were mixed with the substrate, the ester intermediate was immediately produced. In contrast, the R41K, D180N, and D180A mutants formed the intermediate much more slowly than the wild-type enzyme, indicating that Arg41 and Asp180 participate in the formation of the ester intermediate. This study presents a new method to analyze the roles of amino acid residues in catalysis.     

Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and structural comparison with D- and L-2-haloacid dehalogenases.

DL-2-Haloacid dehalogenase from Pseudomonas sp. strain 113 (DL-DEX) catalyzes the hydrolytic dehalogenation of both D- and L-2-haloalkanoic acids to produce the corresponding L- and D-2-hydroxyalkanoic acids, respectively, with inversion of the C2 configuration. DL-DEX is a unique enzyme: it acts on the chiral carbon of the substrate and uses both enantiomers as equivalent substrates. We have isolated and sequenced the gene encoding DL-DEX. The open reading frame consists of 921 bp corresponding to 307 amino acid residues. No sequence similarity between DL-DEX and L-2-haloacid dehalogenases was found. However, DL-DEX had significant sequence similarity with D-2-haloacid dehalogenase from Pseudomonas putida AJ1, which specifically acts on D-2-haloalkanoic acids: 23% of the total amino acid residues of DL-DEX are conserved. We mutated each of the 26 residues with charged and polar side chains, which are conserved between DL-DEX and D-2-haloacid dehalogenase. Thr65, Glu69, and Asp194 were found to be essential for dehalogenation of not only the D- but also the L-enantiomer of 2-haloalkanoic acids. Each of the mutant enzymes, whose activities were lower than that of the wild-type enzyme, acted on both enantiomers of 2-haloacids as equivalent substrates in the same manner as the wild-type enzyme. We also found that each enantiomer of 2-chloropropionate competitively inhibits the enzymatic dehalogenation of the other. These results suggest that DL-DEX has a single and common catalytic site for both enantiomers.     

DL-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a new class of dehalogenase catalyzing hydrolytic dehalogenation not involving enzyme-substrate ester intermediate.

DL-2-Haloacid dehalogenase from Pseudomonas sp. 113 (DL-DEX 113) catalyzes the hydrolytic dehalogenation of D- and L-2-haloalkanoic acids, producing the corresponding L- and D-2-hydroxyalkanoic acids, respectively. Every halidohydrolase studied so far (L-2-haloacid dehalogenase, haloalkane dehalogenase, and 4-chlorobenzoyl-CoA dehalogenase) has an active site carboxylate group that attacks the substrate carbon atom bound to the halogen atom, leading to the formation of an ester intermediate. This is subsequently hydrolyzed, resulting in the incorporation of an oxygen atom of the solvent water molecule into the carboxylate group of the enzyme. In the present study, we analyzed the reaction mechanism of DL-DEX 113. When a single turnover reaction of DL-DEX 113 was carried out with a large excess of the enzyme in H(2)(18)O with a 10 times smaller amount of the substrate, either D- or L-2-chloropropionate, the major product was found to be (18)O-labeled lactate by ionspray mass spectrometry. After a multiple turnover reaction in H(2)(18)O, the enzyme was digested with trypsin or lysyl endopeptidase, and the molecular masses of the peptide fragments were measured with an ionspray mass spectrometer. No peptide fragments contained (18)O. These results indicate that the H(2)(18)O of the solvent directly attacks the alpha-carbon of 2-haloalkanoic acid to displace the halogen atom. This is the first example of an enzymatic hydrolytic dehalogenation that proceeds without producing an ester intermediate.     

2-卤代酸脱卤酶研究进展

2-卤代酸脱卤酶(EC 3.8.1.X)催化2-卤代酸脱卤水解形成相应的2-羟基酸。该类酶不仅能够降解环境中的卤代污染物,而且具有宽广底物谱和高效手性拆分特性,因而在环保和手性中间体的绿色合成中具有广阔应用前景。目前已经对多种2-卤代酸脱卤酶进行生化特性表征,并对酶分子三维结构及催化机制进行了深入研究。文中从2-卤代酸脱卤酶的来源、蛋白质结构与催化反应机制、催化特性及应用方面等研究取得的新进展进行综述,并展望了2-卤代酸脱卤酶的进一步研究方向。     

A Mechanistic Analysis of Enzymatic Degradation of Organohalogen Compounds

Enzymes that catalyze the conversion of organohalogen compounds have been attracting a great deal of attention, partly because of their possible applications in environmental technology and the chemical industry. We have studied the mechanisms of enzymatic degradation of various organic halo acids. In the reaction of L-2-haloacid dehalogenase and fluoroacetate dehalogenase, the carboxylate group of the catalytic aspartate residue nucleophilically attacked the α-carbon atom of the substrates to displace the halogen atom. In the reaction catalyzed by DL-2-haloacid dehalogenase, a water molecule directly attacked the substrate to displace the halogen atom. In the course of studies on the metabolism of 2-chloroacrylate, we discovered two new enzymes. 2-Haloacrylate reductase catalyzed the asymmetric reduction of 2-haloacrylate to produce L-2-haloalkanoic acid in an NADPH-dependent manner. 2-Haloacrylate hydratase catalyzed the hydration of 2-haloacrylate to produce pyruvate. The enzyme is unique in that it catalyzes the non-redox reaction in an FADH₂-dependent manner.     

Purification and characterization of thermostable and nonthermostable 2-haloacid dehalogenases with different stereospecificities from Pseudomonas sp. strain YL.

Two novel hydrolytic dehalogenases, thermostable L-2-haloacid dehalogenase (L-DEX) inducibly synthesized by 2-chloropropionate (2-CPA) and nonthermostable DL-2-haloacid dehalogenase (DL-DEX) induced by 2-chloroacrylate, were purified to homogeneity from Pseudomonas sp. strain YL. DL-DEX consisted of a monomer with a molecular weight of about 36,000 and catalyzed the dehalogenation of L and D isomers of 2-CPA to produce D- and L-lactates, respectively. It acted on 2-haloalkanoic acids with a carbon chain length of 2 to 4. The maximum activity on DL-2-CPA was found at pH 10.5 and 45 degrees C. L-DEX, composed of two subunits with identical molecular weights of 27,000, catalyzes the dehalogenation of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids. The enzyme acts not only on short-carbon-chain 2-haloacids such as monochloroacetate and monoiodoacetate in aqueous solution but also on long-carbon-chain 2-haloacids such as 2-bromohexadecanoate in n-heptane. L-DEX is thermostable: it retained its full activity upon heating at 60 degrees C for 30 min. The pH and temperature optima for dehalogenation of L-2-CPA were 9.5 and 65 degrees C, respectively. L-DEX was strongly inhibited by modification of carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and Woodward reagent K, but DL-DEX was not.     

X-ray structure and mechanism of ZgHAD,a l-2-haloacid dehalogenase from the marine Flavobacterium Zobellia galactanivorans

Haloacid dehalogenases are potentially involved in bioremediation of contaminated environments and few have been biochemically characterized from marine organisms. The l -2-haloacid dehalogenase (l -2-HAD) from the marine Bacteroidetes Zobellia galactanivorans Dsij^T (ZgHAD) has been shown to catalyze the dehalogenation of C2 and C3 short-chain l -2-haloalkanoic acids. To better understand its catalytic properties, its enzymatic stability, active site, and 3D structure were analyzed. ZgHAD demonstrates high stability to solvents and a conserved catalytic activity when heated up to 60°C, its melting temperature being at 65°C. The X-ray structure of the recombinant enzyme was solved by molecular replacement. The enzyme folds as a homodimer and its active site is very similar to DehRhb, the other known l -2-HAD from a marine Rhodobacteraceae. Marked differences are present in the putative substrate entrance sites of the two enzymes. The H179 amino acid potentially involved in the activation of a catalytic water molecule was confirmed as catalytic amino acid through the production of two inactive site-directed mutants. The crystal packing of 13 dimers in the asymmetric unit of an active-site mutant, ZgHAD-H179N, reveals domain movements of the monomeric subunits relative to each other. The involvement of a catalytic His/Glu dyad and substrate binding amino acids was further confirmed by computational docking. All together our results give new insights into the catalytic mechanism of the group of marine l -2-HAD.     

Reactivity of asparagine residue at the active site of the D105N mutant of fluoroacetate dehalogenase from Moraxella sp. B

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon–fluorine bond of fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the α-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the l-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation.     

Purification and properties of a new enzyme, DL-2-haloacid dehalogenase, from Pseudomonas sp.

A new enzyme, DL-2-haloacid dehalogenase, was isolated and purified to homogeneity from the cells of Pseudomonas sp. strain 113. This enzyme catalyzed non-stereospecific dehalogenation of both of the optical isomers of 2-chloropropionate through an SN2 type of reaction; L- and D-lactates were formed from D- and L-2-chloropropionates, respectively. The enzyme acted on 2-halogenated aliphatic carboxylic acids whose carbon chain lengths were less than five. It also dehalogenated trichloroacetate to form oxalate and showed maximum activity at pH 9.5. The Michaelis constants for substrates were as follows: 5.0 mM for monochloroacetate, 1.1 mM for L-2-chloropropionate, and 4.8 mM for D-2-chloropropionate. DL-2-Haloacid dehalogenase was inhibited by HgCl2, ZnSO4, and MnSO4, but was not affected by thiol reagents, such as p-chloromercuribenzoate and iodoacetamide. This enzyme had a molecular weight of about 68,000 and appeared to be composed of two subunits identical in molecular weight.     

Crystal structures of intermediates in the dehalogenation of haloalkanoates by L-2-haloacid dehalogenase.

The L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoates to their corresponding D-2-hydroxyalkanoates, with inversion of the configuration at the C(2) atom. The structure of the apoenzyme at pH 8 was refined at 1.5-A resolution. By lowering the pH, the catalytic activity of the enzyme was considerably reduced, allowing the crystal structure determination of the complexes with L-2-monochloropropionate and monochloroacetate at 1.7 and 2.1 A resolution, respectively. Both complexes showed unambiguous electron density extending from the nucleophile Asp(8) to the C(2) atom of the dechlorinated substrates corresponding to a covalent enzyme-ester reaction intermediate. The halide ion that is cleaved off is found in line with the Asp(8) Odelta1-C(2) bond in a halide-stabilizing cradle made up of Arg(39), Asn(115), and Phe(175). In both complexes, the Asp(8) Odelta2 carbonyl oxygen atom interacts with Thr(12), Ser(171), and Asn(173), which possibly constitute the oxyanion hole in the hydrolysis of the ester bond. The carboxyl moiety of the substrate is held in position by interactions with Ser(114), Lys(147), and main chain NH groups. The L-2-monochloropropionate CH(3) group is located in a small pocket formed by side chain atoms of Lys(147), Asn(173), Phe(175), and Asp(176). The size and position of the pocket explain the stereospecificity and the limited substrate specificity of the enzyme. These crystallographic results demonstrate that the reaction of the enzyme proceeds via the formation of a covalent enzyme-ester intermediate at the nucleophile Asp(8).     

Mechanism of the reaction catalyzed by DL-2-haloacid dehalogenase as determined from kinetic isotope effects

dl-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a unique enzyme because it acts on the chiral carbons of both enantiomers, although its amino acid sequence is similar only to that of d-2-haloacid dehalogenase from Pseudomonas putida AJ1 that specifically acts on (R)-(+)-2-haloalkanoic acids. Furthermore, the catalyzed dehalogenation proceeds without formation of an ester intermediate; instead, a water molecule directly attacks the alpha-carbon of the 2-haloalkanoic acid. We have studied solvent deuterium and chlorine kinetic isotope effects for both stereoisomeric reactants. We have found that chlorine kinetic isotope effects are different: 1.0105 +/- 0.0001 for (S)-(-)-2-chloropropionate and 1.0082 +/- 0.0005 for the (R)-(+)-isomer. Together with solvent deuterium isotope effects on V(max)/K(M), 0.78 +/- 0.09 for (S)-(-)-2-chloropropionate and 0.90 +/- 0.13 for the (R)-(+)-isomer, these values indicate that in the case of the (R)-(+)-reactant another step preceding the dehalogenation is partly rate-limiting. Under the V(max) conditions, the corresponding solvent deuterium isotope effects are 1.48 +/- 0.10 and 0.87 +/- 0.27, respectively. These results indicate that the overall reaction rates are controlled by different steps in the catalysis of (S)-(-)- and (R)-(+)-reactants.     

Purification and characterization of D-2-haloacid dehalogenase from Pseudomonas putida strain AJ1/23

A D-2-haloacid dehalogenase was isolated and purified to homogeneity from Pseudomonas putida strain AJ1/23. The enzyme catalysed the stereospecific dehalogenation of the D-isomer of 2-chloropropionate. Using a new ion-chromatograph assay, the enzyme was found to catalyse the dehalogenation of short-chain 2-halocarboxylic acids. Maximum enzyme activity occurred at pH 9.5 and 50 degrees C and the enzyme was insensitive to most -SH reagents. The enzyme has an Mr of about 135,000 and appears to be composed of four subunits of identical Mr.     

A mechanistic analysis of enzymatic degradation of organohalogen compounds

Enzymes that catalyze the conversion of organohalogen compounds have been attracting a great deal of attention, partly because of their possible applications in environmental technology and the chemical industry. We have studied the mechanisms of enzymatic degradation of various organic halo acids. In the reaction of L-2-haloacid dehalogenase and fluoroacetate dehalogenase, the carboxylate group of the catalytic aspartate residue nucleophilically attacked the α-carbon atom of the substrates to displace the halogen atom. In the reaction catalyzed by DL-2-haloacid dehalogenase, a water molecule directly attacked the substrate to displace the halogen atom. In the course of studies on the metabolism of 2-chloroacrylate, we discovered two new enzymes. 2-Haloacrylate reductase catalyzed the asymmetric reduction of 2-haloacrylate to produce L-2-haloalkanoic acid in an NADPH-dependent manner. 2-Haloacrylate hydratase catalyzed the hydration of 2-haloacrylate to produce pyruvate. The enzyme is unique in that it catalyzes the non-redox reaction in an FADH(2)-dependent manner.     

Purification and characterization of a dehalogenase from Pseudomonas stutzeri DEH130 isolated from the marine sponge Hymeniacidon perlevis

2-haloacid dehalogenases are enzymes that are capable of degrading 2-haloacid compounds. These enzymes are produced by bacteria, but so far they have only been purified and characterized from terrestrial bacteria. The present study describes the purification and characterization of 2-haloacid dehalogenase from the marine bacterium Pseudomonas stutzeri DEH130. P. Stutzeri DEH130 contained two kinds of 2-haloacid dehalogenase (designated as Dehalogenase I and Dehalogenase II) as detected in the crude cell extract after ammonium sulfate fractionation. Both enzymes appeared to exhibit stereo-specificity with respect to substrate. Dehalogenase I was a 109.9-kDa enzyme that preferentially utilized D-2-chloropropropionate and had optimum activity at pH 7.5. Dehalogenase II, which preferentially utilized L-2-chloropropionate, was further purified by ion-exchange chromatography and gel filtration. Purified Dehalogenase II appeared to be a dimeric enzyme with a subunit of 26.0-kDa. It had maximum activity at pH 10.0 and a temperature of 40 °C. Its activity was not inhibited by DTT and EDTA, but strongly inhibited by Cu²⁺, Zn²⁺, and Co²⁺. The K _m and V _max for L-2-chloropropionate were 0.3 mM and 23.8 μmol/min/mg, respectively. Its substrate specificity was limited to short chain mono-substituted 2-halocarboxylic acids, with no activity detected toward fluoropropionate and monoiodoacetate. This is the first report on the purification and characterization of 2-haloacid dehalogenase from a marine bacterium.     

R-2-卤代酸脱卤酶的同源模建及底物对映体选择性研究

对来自假单胞菌ZJU26中的R-2-卤代酸脱卤酶(DehI-R)进行同源模建,分析其与底物的相互作用,为解析酶的底物对映体选择性提供理论依据.采用Sybyl中的APM模块首次构建并优化了R-2-卤代酸脱卤酶的三维结构,并用Procheck 验证结构模型的合理性.使用Suflex-Docking模块将结构模型与底物分别进行对接,分析相互之间的作用.序列比对结果显示,R-2-卤代酸脱卤酶与恶臭假单胞菌PP3中DehI的相似性达23.71％.Deh-R模建后的结构与模板很好的吻合.模型比对分析DehI-R中参与催化的残基,除Asn2.03外大部分都比较保守.分子对接结果表明,R-2-氯丙酸和S-2-氯丙酸都可以结合到活性位点上,决定其选择性的是值点Asn203,在RS-2-卤代酸脱卤酶所对应位点的残基为Ala,相比之下,Aan具备较大的空间位阻,从而阻止了S-2-氯丙酸的反应.利用Sybyl中的Biopolymer模块对R-2-卤代酸脱卤酶中的Asn203突变成具有不同空间位阻的Ala、Gly和Gln.突变酶与底物对接结果进一步证实了Asn203位点对R-2-卤代酸脱卤酶的底物对映体选择性作用.     

A model for the effects of primary substrates on the kinetics of reductive dehalogenation

A kinetic model that describes substrate interactions during reductive dehalogenation reactions is developed. This model describes how the concentrations of primary electron-donor and -acceptor substrates affect the rates of reductive dehalogenation reactions. A basic model, which considers only exogenous electron-donor and -acceptor substrates, illustrates the fundamental interactions that affect reductive dehalogenation reaction kinetics. Because this basic model cannot accurately describe important phenomena, such as reductive dehalogenation that occurs in the absence of exogenous electron donors, it is expanded to include an endogenous electron donor and additional electron acceptor reactions. This general model more accurately reflects the behavior that has been observed for reductive dehalogenation reactions. Under most conditions, primary electron-donor substrates stimulate the reductive dehalogenation rate, while primary electron acceptors reduce the reaction rate. The effects of primary substrates are incorporated into the kinetic parameters for a Monod-like rate expression. The apparent maximum rate of reductive dehalogenation (q _{m, ap} ) and the apparent half-saturation concentration (K _ap ) increase as the electron donor concentration increases. The electron-acceptor concentration does not affect q _{m, ap} , but K _ap is directly proportional to its concentration.Definitions for model parameters RX halogenated aliphatic substrate - E-M ⁿ reduced dehalogenase - E-M ⁿ⁺² oxidized dehalogenase - [E-M ⁿ ] steady-state concentration of the reduced dehalogenase (moles of reduced dehalogenase per unit volume) - [E-M ⁿ⁺²] steady-state concentration of the oxidized dehalogenase (moles of reduced dehalogenase per unit volume) - DH₂ primary exogenous electron-donor substrate - A primary exogenous electron-acceptor substrate - A2 second primary exogenous electron-acceptor substrate - X biomass concentration (biomass per unit volume) - f fraction of biomass that is comprised of the dehalogenase (moles of dehalogenase per unit biomass) - stoichiometric coefficient for the reductive dehalogenation reaction (moles of dehalogenase oxidized per mole of halogenated substrate reduced) - stoichiometric coefficient for oxidation of the primary electron donor (moles of dehalogenase reduced per mole of donor oxidized) - stoichiometric coefficient for oxidation of the endogenous electron donor (moles of dehalogenase reduced per unit biomass oxidized) - stoichiometric coefficient for reduction of the primary electron acceptor (moles of dehalogenase oxidized per mole of acceptor reduced) - stoichiometric coefficient for reduction of the second electron acceptor (moles of dehalogenase oxidized per mole of acceptor reduced) - r _RX rate of the reductive dehalogenation reaction (moles of halogenated substrate reduced per unit volume per unit time) - r _d1 rate of oxidation of the primary exogenous electron donor (moles of donor oxidized per unit volume per unit time) - r _d2 rate of oxidation of the endogenous electron donor (biomass oxidized per unit volume per unit time) - r _a1 rate of reduction of the primary exogenous electron acceptor (moles of acceptor reduced per unit volume per unit time) - r _a2 rate of reduction of the second primary electron acceptor (moles of acceptor reduced per unit volume per unit time) - k _RX mixed second-order rate coefficient for the reductive dehalogenation reaction (volume per mole dehalogenase per unit time) - k _d1 mixed-second-order rate coefficient for oxidation of the primary electron donor (volume per mole dehalogenase per unit time) - k _d2 mixed-second-order rate coefficient for oxidation of the endogenous electron donor (volume per mole dehalogenase per unit time) - b first-order biomass decay coefficient (biomass oxidized per unit biomass per unit time) - k _a1 mixed-second-order rate coefficient for reduction of the primary electron acceptor (volume per mole dehalogenase per unit time) - k _a2 mixed-second-order rate coefficient for reduction of the second primary electron acceptor (volume per mole dehalogenase per unit time) - q _m,ap apparent maximum specific rate of reductive dehalogenation (moles of RX per unit biomass per unit time) - K _ap apparent half-saturation concentration for the halogenated aliphatic substrate (moles of RX per unit volume) - k _ap apparent pseudo-first-order rate coefficient for reductive dehalogenation (volume per unit biomass per unit time)     

Crystallization and preliminary X-ray crystallographic studies of L-2-haloacid dehalogenase from Pseudomonas sp. YL

The dimeric L -2-haloacid dehalogenase from Pseudomonas sp. YL, (subunit mass, 26179 Da), has been crystallized by vapor diffusion, supplemented by repetitive seeding, against a 50 mM potassium dihydrogenphosphate solution (pH 4.5) containing 15% (w/v) polyethylene glycol 8,000 and 1% (v/v) n-propanol. The crystals belong to the monoclinic space group C2 with unit cell dimensions of a = 92.21 Å, b = 62.78 Angst; c = 50.84 Å, and β = 122.4°, and contain two dehalogenase dimers in the unit cell. They are of good quality and diffract up to 1.5 Å resolution.     

Bacterial dehalogenases: biochemistry, genetics, and biotechnological applications.

This review is a survey of bacterial dehalogenases that catalyze the cleavage of halogen substituents from haloaromatics, haloalkanes, haloalcohols, and haloalkanoic acids. Concerning the enzymatic cleavage of the carbon-halogen bond, seven mechanisms of dehalogenation are known, namely, reductive, oxygenolytic, hydrolytic, and thiolytic dehalogenation; intramolecular nucleophilic displacement; dehydrohalogenation; and hydration. Spontaneous dehalogenation reactions may occur as a result of chemical decomposition of unstable primary products of an unassociated enzyme reaction, and fortuitous dehalogenation can result from the action of broad-specificity enzymes converting halogenated analogs of their natural substrate. Reductive dehalogenation either is catalyzed by a specific dehalogenase or may be mediated by free or enzyme-bound transition metal cofactors (porphyrins, corrins). Desulfomonile tiedjei DCB-1 couples energy conservation to a reductive dechlorination reaction. The biochemistry and genetics of oxygenolytic and hydrolytic haloaromatic dehalogenases are discussed. Concerning the haloalkanes, oxygenases, glutathione S-transferases, halidohydrolases, and dehydrohalogenases are involved in the dehalogenation of different haloalkane compounds. The epoxide-forming halohydrin hydrogen halide lyases form a distinct class of dehalogenases. The dehalogenation of alpha-halosubstituted alkanoic acids is catalyzed by halidohydrolases, which, according to their substrate and inhibitor specificity and mode of product formation, are placed into distinct mechanistic groups. beta-Halosubstituted alkanoic acids are dehalogenated by halidohydrolases acting on the coenzyme A ester of the beta-haloalkanoic acid. Microbial systems offer a versatile potential for biotechnological applications. Because of their enantiomer selectivity, some dehalogenases are used as industrial biocatalysts for the synthesis of chiral compounds. The application of dehalogenases or bacterial strains in environmental protection technologies is discussed in detail.     

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

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 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 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 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.Dehalogenases catalyze the removal of halogen atoms from organohalogen compounds. These enzymes have been attracting a great deal of attention partly because of their possible applications to the chemical industry and environmental technology. Several dehalogenases have been discovered and characterized (6, 11, 14, 17, 22). Some of them act on unsaturated aliphatic organohalogen compounds in which a halogen atom is bound to an sp²-hybridized carbon atom. Examples include various corrinoid/iron-sulfur cluster-containing reductive dehalogenases (1, 7), cis- and trans-3-chloroacrylic acid dehalogenases (4, 19), and LinF (maleylacetate reductase), which acts on 2-chloromaleylacetate (5).In order to gain more insight into the enzymatic dehalogenation of unsaturated aliphatic organohalogen compounds, we searched for microorganisms that dissimilate 2-chloroacrylate (2-CAA) as a sole source of carbon and energy (8). 2-CAA is a bacterial metabolite of 2-chloroallyl alcohol, an intermediate or by-product in the industrial synthesis of herbicides (26). Rats treated orally with the herbicides sulfallate, diallate, and triallate excrete urinary 2-CAA (16). Various halogenated acrylic acids are produced by a red alga (27). We obtained three 2-CAA-utilizing bacteria as a result of screening (8). 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.Another bacterium that we obtained, Pseudomonas sp. strain YL, also dissimilates 2-CAA. However, the metabolic fate of 2-CAA in this bacterium remains unclear. In the present study, we analyzed proteins from 2-CAA- and lactate-grown cells of Pseudomonas sp. YL by two-dimensional polyacrylamide gel electrophoresis (PAGE) and identified a 2-CAA-inducible protein. We found that the protein catalyzes the dehalogenation of 2-CAA by the addition of a water molecule to the substrate, representing a new family of dehalogenases that act on unsaturated aliphatic organohalogen compounds. Remarkably, the enzyme requires reduced flavin adenine dinucleotide (FAD) (FADH₂) for its activity, although the reaction does not involve a net change in the redox state of the coenzyme or substrate. Here we describe the occurrence and characteristics of this unusual flavoenzyme.     

Biochemical and structural studies of a l-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii

Haloacid dehalogenases have potential applications in the pharmaceutical and fine chemical industry as well as in the remediation of contaminated land. The l-2-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned and over-expressed in Escherichia coli and successfully purified to homogeneity. Here we report the structure of the recombinant dehalogenase solved by molecular replacement in two different crystal forms. The enzyme is a homodimer with each monomer being composed of a core-domain of a β-sheet bundle surrounded by α-helices and an α-helical sub-domain. This fold is similar to previously solved mesophilic l-haloacid dehalogenase structures. The monoclinic crystal form contains a putative inhibitor l-lactate in the active site. The enzyme displays haloacid dehalogenase activity towards carboxylic acids with the halide attached at the C2 position with the highest activity towards chloropropionic acid. The enzyme is thermostable with maximum activity at 60°C and a half-life of over 1 h at 70°C. The enzyme is relatively stable to solvents with 25% activity lost when incubated for 1 h in 20% v/v DMSO.