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Several pathways of biotic dechlorination can be found in enzymes, each characterized by different chlorine isotopic fractionation, which can thus serve as a signature of a particular mechanism. Unlike other dehalogenases, DL-2-haloacid dehalogenase, DL-DEX, converts both enantiomers of the substrate. Chlorine isotope effects for this enzyme are larger than in the case of other dehalogenases. Recently, the 3D structure of this enzyme became available and enabled us to model these isotope effects and seek their origin. We show that the elevated values of the chlorine kinetic isotope effects originate in part in the processes of binding and migration within the enzyme active site that precede the dehalogenation step., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The high substrate specificity of fluoroacetate dehalogenase was explored by using crystallographic analysis, fluorescence spectroscopy, and theoretical computations. A crystal structure for the Asp104Ala mutant of the enzyme from Burkholderia sp. FA1 complexed with fluoroacetate was determined at 1.2 Å resolution. The orientation and conformation of bound fluoroacetate is different from those in the crystal structure of the corresponding Asp110Asn mutant of the enzyme from Rhodopseudomonas palustris CGA009 reported recently (J. Am. Chem. Soc. 2011, 133, 7461). The fluorescence of the tryptophan residues of the wild-type and Trp150Phe mutant enzymes from Burkholderia sp. FA1 incubated with fluoroacetate and chloroacetate was measured to gain information on the environment of the tryptophan residues. The environments of the tryptophan residues were found to be different between the fluoroacetate- and chloroacetate-bound enzymes; this would come from different binding modes of these two substrates in the active site. Docking simulations and QM/MM optimizations were performed to predict favorable conformations and orientations of the substrates. The F atom of the substrate is oriented toward Arg108 in the most stable enzyme-fluoroacetate complex. This is a stable but unreactive conformation, in which the small O-C-F angle is not suitable for the S(N)2 displacement of the F(-) ion. The cleavage of the C-F bond is initiated by the conformational change of the substrate to a near attack conformation (NAC) in the active site. The second lowest energy conformation is appropriate for NAC; the C-O distance and the O-C-F angle are reasonable for the S(N) 2 reaction. The activation energy is greatly reduced in this conformation because of three hydrogen bonds between the leaving F atom and surrounding amino acid residues. Chloroacetate cannot reach the reactive conformation, due to the longer C-Cl bond; this results in an increase of the activation energy despite the weaker C-Cl bond., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

L-Azetidine-2-carboxylate hydrolase from Pseudomonas sp. strain A2C catalyzes a ring-opening reaction that detoxifies L-azetidine-2-carboxylate, an analogue of L-proline. Recombinant L-azetidine-2-carboxylate hydrolase was overexpressed, purified and crystallized using polyethylene glycol and magnesium acetate as precipitants. The needle-shaped crystal belonged to space group P2(1), with unit-cell parameters a = 35.6, b = 63.6, c = 54.7 A, beta = 105.5 degrees . The crystal diffracted to a resolution of 1.38 A. The calculated V(M) value was 2.2 A(3) Da(-1), suggesting that the crystal contains one enzyme subunit in the asymmetric unit.

L-2-haloacid dehalogenase (L-DEX) catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids. This enzyme is expected to be applicable to the bioremediation of environments contaminated with halogenated organic compounds. We analyzed the reaction mechanism of L-DEX from Pseudomonas sp. YL (L-DEX YL) by using molecular modeling. The complexes of wild-type L-DEX YL and its K151A and D180A mutants with its typical substrate, L-2-chloropropionate, were constructed by docking simulation. Subsequently, molecular dynamics (MD) and ab initio fragment molecular orbital (FMO) calculations of the complexes were performed. The ab initio FMO method was applied at the MP2/6-31G level to estimate interfragment interaction energies. K151 and D180, which are experimentally shown to be important for enzyme activity, interact particularly strongly with L-2-chloropropionate, catalytic water, nucleophile (D10), and with each other. Our calculations suggest that K151 stabilizes substrate orientation and balances the charge around the active site, while D180 stabilizes the rotation of the nucleophile D10, fixes catalytic water around D10, and prevents K151 from approaching D10. Further, D180 may activate catalytic water on its own or with K151, S175, and N177. These roles are consistent with the previous results. Thus, MD and ab initio FMO calculations are powerful tools for the elucidation of the mechanism of enzymatic reaction at the molecular level and can be applied to other catalytically important residues. The results obtained here will play an important role in elucidating the reaction mechanism and rational design of L-DEX YL with improved enzymatic activity or substrate specificity., ((c) 2009 Wiley Periodicals, Inc.)

The biological dehalogenation of fluoroacetate carried out by fluoroacetate dehalogenase is discussed by using quantum mechanical/molecular mechanical (QM/MM) calculations for a whole-enzyme model of 10 800 atoms. Substrate fluoroacetate is anchored by a hydrogen-bonding network with water molecules and the surrounding amino acid residues of Arg105, Arg108, His149, Trp150, and Tyr212 in the active site in a similar way to haloalkane dehalogenase. Asp104 is likely to act as a nucleophile to attack the alpha-carbon of fluoroacetate, resulting in the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule to the carbonyl carbon atom. The cleavage of the strong C-F bond is greatly facilitated by the hydrogen-bonding interactions between the leaving fluorine atom and the three amino acid residues of His149, Trp150, and Tyr212. The hydrolysis of the ester intermediate is initiated by a proton transfer from the water molecule to His271 and by the simultaneous nucleophilic attack of the water molecule. The transition state and produced tetrahedral intermediate are stabilized by Asp128 and the oxyanion hole composed of Phe34 and Arg105.

Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of a carbon-fluorine bond of an aliphatic compound: the bond energy of the carbon-fluorine bond is among the highest found in natural products. The enzyme also acts on chloroacetate, although much less efficiently. We here determined the X-ray crystal structure of the enzyme from Burkholderia sp. strain FA1 as the first experimentally determined three-dimensional structure of fluoroacetate dehalogenase. The enzyme belongs to the alpha/beta hydrolase superfamily and exists as a homodimer. Each subunit consists of core and cap domains. The catalytic triad, Asp104-His271-Asp128, of which Asp104 serves as the catalytic nucleophile, was found in the core domain at the domain interface. The active site was composed of Phe34, Asp104, Arg105, Arg108, Asp128, His271, and Phe272 of the core domain and Tyr147, His149, Trp150, and Tyr212 of the cap domain. An electron density peak corresponding to a chloride ion was found in the vicinity of the N(epsilon1) atom of Trp150 and the N(epsilon2) atom of His149, suggesting that these are the halide ion acceptors. Site-directed replacement of each of the active-site residues, except for Trp150, by Ala caused the total loss of the activity toward fluoroacetate and chloroacetate, whereas the replacement of Trp150 caused the loss of the activity only toward fluoroacetate. An interaction between Trp150 and the fluorine atom is probably an absolute requirement for the reduction of the activation energy for the cleavage of the carbon-fluorine bond.

Dehalogenases catalyze the cleavage of the carbon-halogen bond of organohalogen compounds. They have been attracting a great deal of attention partly because of their potential applications in the chemical industry and bioremediation. In this personal account, we describe occurrences, reaction mechanisms, and applications of bacterial hydrolytic dehalogenases and related enzymes, particularly L-2-haloacid dehalogenase, DL-2-haloacid dehalogenase, fluoroacetate dehalogenase, and 2-haloacrylate reductase. L-2-Haloacid dehalogenase is a representative enzyme of the haloacid dehalogenase (HAD) superfamily, which includes the P-type ATPases and other hydrolases. Structural and mechanistic analyses of this enzyme have yielded important insights into the mode of action of the HAD superfamily proteins. Fluoroacetate dehalogenase is unique in that it catalyzes the cleavage of the highly stable C--F bond of a fluorinated aliphatic compound. In the reactions of L-2-haloacid dehalogenase and fluoroacetate dehalogenase, the carboxylate group of Asp performs a nucleophilic attack on the alpha-carbon atom of the substrate, displacing the halogen atom. This mechanism is common to haloalkane dehalogenase and 4-chlorobenzoyl-CoA dehalogenase. DL-2-Haloacid dehalogenase is unique in that a water molecule directly attacks the substrate, displacing the halogen atom. The occurrence of 2-haloacrylate reductase was recently reported, revealing a new pathway for the degradation of unsaturated aliphatic organohalogen compounds., (Copyright 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.)

DL-2-Haloacid dehalogenase from Methylobacterium sp. CPA1 (DL-DEX Mb) is a unique enzyme that catalyzes the dehalogenation reaction without the formation of an ester intermediate. A recombinant form of DL-DEX Mb has been expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the hexagonal space group P6(3), with unit-cell parameters a = b = 186.2, c = 114.4 A. The crystals are likely to contain between four and eight monomers in the asymmetric unit, with a V(M) value of 4.20-2.10 A3 Da(-1). A self-rotation function revealed peaks on the chi = 180 degrees section. X-ray data have been collected to 1.75 A resolution.

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.

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 alpha-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.

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes the hydrolytic dehalogenation of fluoroacetate and other haloacetates. Asp(105) of the enzyme acts as a nucleophile to attack the alpha-carbon of haloacetate to form an ester intermediate, which is subsequently hydrolyzed by a water molecule activated by His(272) [Liu, J.Q., Kurihara, T., Ichiyama, S., Miyagi, M., Tsunasawa, S., Kawasaki, H., Soda, K., and Esaki, N. (1998) J. Biol. Chem. 273, 30897-30902]. In this study, we found that FAc-DEX is inactivated concomitantly with defluorination of fluoroacetate by incubation with ammonia. Mass spectrometric analyses revealed that the inactivation of FAc-DEX is caused by nucleophilic attack of ammonia on the ester intermediate to convert the catalytic residue, Asp(105), into an asparagine residue. The results indicate that ammonia reaches the active site of FAc-DEX without losing its nucleophilicity. Analysis of the three-dimensional structure of the enzyme by homology modeling showed that the active site of the enzyme is mainly composed of hydrophobic and basic residues, which are considered to be essential for an ammonia molecule to retain its nucleophilicity. In a normal enzyme reaction, the hydrophobic environment is supposed to prevent hydration of the highly electronegative fluorine atom of the substrate and contribute to fluorine recognition by the enzyme. Basic residues probably play a role in counterbalancing the electronegativity of the substrate. These results demonstrate that catalysis-linked inactivation is useful for characterizing the active-site environment as well as for identifying the catalytic residue.

l-2-Haloacid dehalogenase from Pseudomonas sp. YL catalyzes the hydrolytic dehalogenation, in which Asp(10) acts as a nucleophile to attack the alpha-carbon of l-2-haloalkanoates to form an ester intermediate, which is subsequently hydrolyzed to produce d-2-hydroxyalkanoates. Surprisingly, replacement of the catalytic residue, Asp(10), by Asn did not result in total inactivation of the enzyme (Kurihara, T., Liu, J.-Q., Nardi-Dei, V., Koshikawa, H., Esaki, N., and Soda, K. (1995) J. Biochem. 117, 1317-1322). In this study, we monitored the D10N mutant enzyme reaction by ion-spray mass spectrometry, and found that the enzyme shows a unique structural change when it was incubated with the substrate, l-2-chloropropionate. LC/MS and tandem MS/MS analyses revealed that Asn(10) attacks the substrate to form an imidate, and a proton and d-lactic acid are eliminated to produce a nitrile (beta-cyanoalanine residue), followed by hydrolysis to reproduce Asn(10). This is the first report of the function of Asn to catalyze nucleophilic substitution through its conversion to beta-cyanoalanine residue as an intermediate structure. Also, these results demonstrate that mass spectrometry is remarkably useful in monitoring enzyme reactions.

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.

Fluoroacetate dehalogenase (EC 3.8.1.3) catalyzes the dehalogenation of fluoroacetate and other haloacetates. The amino acid sequence of fluoroacetate dehalogenase from Moraxella sp. B is similar to that of haloalkane dehalogenase (EC 3.8.1.5) from Xanthobacter autotrophicus GJ10 in the regions around Asp-105 and His-272, which correspond to the active site nucleophile Asp-124 and the base catalyst His-289 of the haloalkane dehalogenase, respectively (Krooshof, G. H., Kwant, E. M., Damborský, J., Koca, J., and Janssen, D. B. (1997) Biochemistry 36, 9571-9580). After multiple turnovers of the fluoroacetate dehalogenase reaction in H218O, the enzyme was digested with trypsin, and the molecular masses of the peptide fragments formed were measured by ion-spray mass spectrometry. Two 18O atoms were shown to be incorporated into the octapeptide, Phe-99-Arg-106. Tandem mass spectrometric analysis of this peptide revealed that Asp-105 was labeled with two 18O atoms. These results indicate that Asp-105 acts as a nucleophile to attack the alpha-carbon of the substrate, leading to the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule on the carbonyl carbon atom. A His-272 --> Asn mutant (H272N) showed no activity with either fluoroacetate or chloroacetate. However, ion-spray mass spectrometry revealed that the H272N mutant enzyme was covalently alkylated with the substrate. The reaction of the H272N mutant enzyme with [14C]chloroacetate also showed the incorporation of radioactivity into the enzyme. These results suggest that His-272 probably acts as a base catalyst for the hydrolysis of the covalent ester intermediate.

The crystal structure of a complex prepared by soaking a single crystal of the S175A mutant of L-2-haloacid dehalogenase from Pseudomonas sp. YL in a solution containing a poor substrate, L-2-chloropropionamide, has been determined by X-ray analysis at 2. 15 A resolution with a crystallographic R factor of 19.8%. The present analysis has revealed the structure of the reaction intermediate trapped in the crystal. In the intermediate, the substrate moiety lacking chlorine is covalently bound to the carboxyl group of D10, and adopts the pro-D-configuration at the C2 atom. The amide group of the substrate is hydrogen-bonded with the hydroxy group of S118. The methyl group bound to the C2 atom exists in a hydrophobic pocket which is important for recognition of the alkyl group of the substrate. The guanidino group of R41 has reasonable orientation for halogen-abstraction.

Crystal structures of L-2-haloacid dehalogenase from Pseudomonas sp. YL complexed with monochloroacetate, L-2-chlorobutyrate, L-2-chloro-3-methylbutyrate, or L-2-chloro-4-methylvalerate were determined at 1.83-, 2.0-, 2.2-, and 2.2-A resolutions, respectively, using the complex crystals prepared with the S175A mutant, which are isomorphous with those of the wild-type enzyme. These structures exhibit unique structural features that correspond to those of the reaction intermediates. In each case, the nucleophile Asp-10 is esterified with the dechlorinated moiety of the substrate. The substrate moieties in all but the monochloroacetate intermediate have a D-configuration at the C2 atom. The overall polypeptide fold of each of the intermediates is similar to that of the wild-type enzyme. However, it is clear that the Asp-10-Ser-20 region moves to the active site in all of the intermediates, and the Tyr-91-Asp-102 and Leu-117-Arg-135 regions make conformational changes in all but the monochloroacetate intermediates. Ser-118 is located near the carboxyl group of the substrate moiety; this residue probably serves as a binding residue for the substrate carboxyl group. The hydrophobic pocket, which is primarily composed of the Tyr-12, Gln-42, Leu-45, Phe-60, Lys-151, Asn-177, and Trp-179 side chains, exists around the alkyl group of the substrate moiety. This pocket may play an important role in stabilizing the alkyl group of the substrate moiety through hydrophobic interactions, and may also play a role in determining the stereospecificity of the enzyme. Moreover, a water molecule, which is absent in the substrate-free enzyme, is present in the vicinities of the carboxyl carbon of Asp-10 and the side chains of Asp-180, Asn-177, and Ala-175 in each intermediate. This water molecule may hydrolyze the ester intermediate and its substrate. These findings crystallographically demonstrate that the enzyme reaction proceeds through the formation of an ester intermediate with the enzyme's nucleophile Asp-10.

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.

Asp10 of L-2-haloacid dehalogenase from Pseudomonas sp. YL was proposed to act as a nucleophile to attack the alpha-carbon of L-2-haloalkanoic acids to form an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon (Liu, J.-Q, Kurihara, T., Miyagi, M., Esaki, N., and Soda, K. (1995) J. Biol. Chem. 270, 18309-18312). We have found that the enzyme is paracatalytically inactivated by hydroxylamine in the presence of the substrates monochloroacetate and L-2-chloropropionate. Ion spray mass spectrometry demonstrated that the molecular mass of the enzyme inactivated by hydroxylamine during the dechlorination of monochloroacetate is about 74 Da greater than that of the native enzyme. To determine the increase of the molecular mass more precisely, we digested the inactivated enzyme with lysyl endopeptidase and measured the molecular masses of the peptide fragments. The molecular mass of the hexapeptide Gly6-Lys11 was shown to increase by 73 Da. Tandem mass spectrometric analysis of this peptide revealed that the increase is due to a modification of Asp10. When the enzyme was paracatalytically inactivated by hydroxylamine during the dechlorination of L-2-chloropropionate, the molecular mass of the hexapeptide was 87 Da higher. Hydroxylamine is proposed to attack the carbonyl carbon of the ester intermediate and form a stable aspartate beta-hydroxamate carboxyalkyl ester residue in the inactivated enzyme.

L-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. The crystal structure of the homodimeric enzyme from Pseudomonas sp. YL has been determined by a multiple isomorphous replacement method and refined at 2.5 A resolution to a crystallographic R-factor of 19.5%. The subunit consists of two structurally distinct domains: the core domain and the subdomain. The core domain has an alpha/beta structure formed by a six-stranded parallel beta-sheet flanked by five alpha-helices. The subdomain inserted into the core domain has a four helix bundle structure providing the greater part of the interface for dimer formation. There is an active site cavity between the domains. An experimentally identified nucleophilic residue, Asp-10, is located on a loop following the amino-terminal beta-strand in the core domain, and other functional residues, Thr-14, Arg-41, Ser-118, Lys-151, Tyr-157, Ser-175, Asn-177, and Asp-180, detected by a site-directed mutagenesis experiment, are arranged around the nucleophile in the active site. Although the enzyme is an alpha/beta-type hydrolase, it does not belong to the alpha/beta hydrolase fold family, from the viewpoint of the topological feature and the position of the nucleophile.

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 angstrom, b = 62.78 angstrom, c = 50.84 angstrom, and beta = 122.4 degrees, and contain two dehalogenase dimers in the unit cell. They are of good quality and diffract up to 1.5 angstrom resolution.

The gene encoding thermostable L-2-halo acid dehalogenase of Pseudomonas sp. YL was isolated, and its overexpression system was constructed. Gene library was prepared from Sau3AI fragments of total DNA from Ps. sp. YL, pUC118 as a vector and Escherichia coli JM109 as a host. The recombinant cells resistant to bromoacetate, a germicide, were isolated and shown to produce L-2-halo acid dehalogenase. Subsequently, subcloning was carried out with pKK223-3 as a vector, and the length of DNA inserted was reduced to 1.1 kbp. One of the subclones showed very high activity, and the amount of the dehalogenase produced corresponded to about 30% of the soluble protein. From 5 g (wet weight) of cells, 105 mg of dehalogenase was efficiently purified by heat treatment and DEAE-Toyopearl chromatography. This overexpression system provides a large amount of the thermostable enzyme to enable us to study the properties, structure and application of the enzyme.

L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the hydrolytic dehalogenation of L-2-haloacids to produce the corresponding D-2-hydroxy acids. We have analyzed the reaction mechanism of the enzyme from Pseudomonas sp. YL and found that Asp10 is the active site nucleophile. When the multiple turnover enzyme reaction was carried out in H2(18)O with L-2-chloropropionate as a substrate, lactate produced was labeled with 18O. However, when the single turnover enzyme reaction was carried out by use of a large excess of the enzyme, the product was not labeled. This suggests that an oxygen atom of the solvent water is first incorporated into the enzyme and then transferred to the product. After the multiple turnover reaction in H2(18)O, the enzyme was digested with lysyl endopeptidase, and the molecular masses of the peptide fragments formed were measured by an ionspray mass spectrometer. Two 18O atoms were shown to be incorporated into a hexapeptide, Gly6-Lys11. Tandem mass spectrometric analysis of this peptide revealed that Asp10 was labeled with two 18O atoms. Our previous site-directed mutagenesis experiment showed that the replacement of Asp10 led to a significant loss in the enzyme activity. These results indicate that Asp10 acts as a nucleophile on the alpha-carbon of the substrate leading to the formation of an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon atom.

L-2-Halo acid dehalogenase catalyzes the stereospecific hydrolytic dehalogenation of L-2-halo acids, with inversion of the C2-configuration. Seven L-2-halo acid dehalogenases from various bacterial strains are significantly similar to one another in their amino acid sequences (36-70% identity), and they are supposed to catalyze the reaction through the same mechanism. To identify catalytically important residues, we mutated all the 36 highly conserved charged and polar amino acid residues of L-2-halo acid dehalogenase from Pseudomonas sp. YL, which consists of 232 amino acid residues, by replacement of D by N, E by Q, R by K, and vice versa, S and T by A, Y and W by F, M by L, and H by N. We found that the replacement of D10, K151, S175, D180, R41, S118, T14, Y157, and N177 led to a significant loss in the enzyme activity or an increase in the Km value for the substrate, showing their involvement in the catalysis. The roles of these residues are discussed.

We have determined the nucleotide sequence of the gene encoding thermostable L-2-halo acid dehalogenase (L-DEX) from the 2-chloroacrylate-utilizable bacterium Pseudomonas sp. strain YL. The open reading frame consists of 696 nucleotides corresponding to 232 amino acid residues. The protein molecular weight was estimated to be 26,179, which was in good agreement with the subunit molecular weight of the enzyme. The gene was efficiently expressed in the recombinant Escherichia coli cells: the amount of L-DEX corresponds to about 49% of the total soluble proteins. The predicted amino acid sequence showed a high level of similarity to those of L-DEXs from other bacterial strains and haloacetate dehalogenase H-2 from Moraxella sp. strain B (38 to 57% identity) but a very low level of similarity to those of haloacetate dehalogenase H-1 from Moraxella sp. strain B (10%) and haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (12%). By searching the protein amino acid sequence database, we found two E. coli hypothetical proteins similar to the Pseudomonas sp. strain YL L-DEX (21 to 22%).

His20 of L-2-halo acid dehalogenase from Pseudomonas cepacia MBA4 was suggested to serve as a catalytic base [Biochem. J. (1993) 292, 69-74]. In this study, we substituted Asn or Leu for His19 of L-2-halo acid dehalogenase from Pseudomonas sp. YL, which corresponds to His20 of the P. cepacia enzyme. Although the substrate specificity was affected by the substitution, the susceptibilities of substrate halo acids were not substantially diminished, and the Km and kcat values of the mutant enzymes for L-2-chloropropionate were not significantly different from those of the wild-type enzyme. In addition, the wild-type and mutant enzymes showed the same pH optimum. Accordingly, His19 is not essential for catalysis of L-2-halo acid dehalogenase.

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.