INTRODUCTION

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Halogenated aliphatics constitute an important class of environmental pollutants. Various microorganisms have evolved that are able to degrade some of these compounds and use them as sole sources of carbon and energy. Such organisms are of importance for bioremediation of polluted soil, groundwater, and wastewater. In most cases, specialized enzymes, designated dehalogenases, catalyze the cleavage of the carbon-halogen bonds, which is a key detoxification reaction. Hydrolytic dehalogenases have been studied extensively, which has resulted in detailed insight into the structure and mechanism of several enzymes of this class (8, 33). For other dehalogenases, structural and mechanistic data are hardly available.

Halohydrin dehalogenases, also referred to as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, occur in the degradation pathways of halopropanols and 1,2-dibromoethane, where they catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, yielding an epoxide, a proton, and a halide ion (7, 22, 30, 31). These enzymes also efficiently catalyze the reverse reaction, the halogenation of epoxides, and the dehalogenation of vicinal chlorocarbonyls to hydroxycarbonyls (2, 14, 31). The interest in halohydrin dehalogenases increased when it was found that the dehalogenation of halohydrins may proceed with high enantioselectivity, making these enzymes useful catalysts for the production of optically pure epoxides and halohydrins (1, 14-16).

In this study, we report the cloning of three bacterial halohydrin dehalogenase genes. Sequence analysis suggested that these proteins are similar to proteins of the short-chain dehydrogenase/reductase (SDR) family. The amino acids Ser132, Tyr145, and Arg149 were identified as the catalytic residues and are proposed to play a role highly similar to that of the conserved residues involved in the redox reaction catalyzed by the SDR family proteins.

	MATERIALS AND METHODS

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Materials. All chemicals were purchased from Acros Chimica, Merck, Aldrich, or Sigma. Molecular biology enzymes were purchased from Boehringer. Oligonucleotide primers were supplied by Eurosequence BV, Groningen, The Netherlands.

Strains and growth conditions. Agrobacterium radiobacter strain AD1 and Arthrobacter sp. strain AD2 were maintained on nutrient broth at 30°C. Mycobacterium sp. strain GP1 was maintained on selective plates with 1-propanol or 1,2-dibromoethane as a carbon source as described before (22). Escherichia coli strains HB101 (6), JM101 (35), and BL21(DE3) (28) were grown at 37°C in Luria-Bertani medium. For selection of recombinants carrying plasmids, the appropriate antibiotic was added at the following concentrations: 50 µg ml¹ for kanamycin, 50 µg ml¹ for ampicillin, and 12.5 µg ml¹ for tetracycline. E. coli BL21(DE3) grown at 17°C was used for high-level expression of recombinant halohydrin dehalogenase driven by the T7 promoter in plasmid pGEF+ (25).

Construction and screening of genomic libraries of halohydrin dehalogenase-producing bacteria. Procedures for the isolation and manipulation of DNA were performed essentially as described by Sambrook et al. (24). The construction of a genomic library of Mycobacterium sp. strain GP1 was described before (22). The same procedure was used to construct a genomic library of A. radiobacter strain AD1 in the cosmid vector pLAFR3 (27). Restriction analysis of plasmids isolated from 16 transduced E. coli HB101 clones showed that all plasmids contained inserts. About 1,000 transductants were screened for halohydrin dehalogenase activity by monitoring halide production with 1,3-dichloro-2-propanol as a substrate as described before (22).

PCR and construction of expression vectors for halohydrin dehalogenase genes. For overexpression, the halohydrin dehalogenase genes were amplified by PCR under conditions described before (32). The hheC gene was amplified from a plasmid preparation of pAD1-9B2 with the forward primer PFHheC (5'-ATCTGACCATGGCAACCGCAATTG-3') and the reverse primer PRHheC (5'-CCCAACGGATCCACGAACCACGGC-3') (NcoI and BamHI sites underlined, start codon shown in boldface, and substituted nucleotides shown in italics). The hheB_GP1 gene starting with the second possible start codon was amplified from recombinant cosmid pGP1-4B5 (22) with the forward primer PF1HheB_GP1 (5'-AAAACCATGGCTAACGGAAGACTGGCAGGC-3') and reverse primer PRHheB_GP1 (5'-GGGCTGTGGATCCTCTCAGGTGGCCCAGCCGCC-3'). The hheA_AD2 gene was directly amplified from cells of Arthrobacter sp. strain AD2 with the forward primer PFHheA_AD2 (5'-GAACCATGGTGATCGCCCTCGTGAC-3') and the reverse primer PRHheA_AD2 (5'-TGGCTATCTGCCCTAACCATGGCC-3'). The hheA_AD2 gene was cloned behind the T7 promoter in the NcoI site, and hheC and hheB_GP1 were cloned between the NcoI and BamHI sites of the expression vector pGEF+ (25).

Nucleotide sequencing. Sequencing on double-stranded DNA was performed with the Amersham Thermo Sequenase cycle sequencing kit (Amersham BV, Roosendaal, The Netherlands), with 7-deaza-dGTP and 5' Cy5 fluorescent primers. Sequence reactions were run on the Pharmacia ALF-Express automatic sequencing machine (Uppsala, Sweden) at the BioMedical Technology Centre (Academic Hospital, Groningen, The Netherlands). Both strands were sequenced to ensure accuracy.

Homology searches and structure prediction. The BLAST program was used to screen DNA and protein databases for similar proteins. Multiple sequence alignments were made in ClustalW v1.7. Secondary structures were predicted with the programs SopM (10) and SSP (26). Tertiary structure modeling was done by comparative protein modeling to known three-dimensional structures of members of the SDR protein family with the program SWISS-MODEL (12) by using the structures of 2HSD, 1AE1, 2AE1, 1AHH, and 1A4U (Protein Data Base codes) as templates.

Overexpression and purification of the halohydrin dehalogenases. Both wild-type and mutant halohydrin dehalogenase genes were expressed in E. coli BL21(DE3) as described before (23). A 1-liter culture of E. coli BL21(DE3) (pGEFHheC) was harvested by centrifugation for purification of HheC. Cells were resuspended in 10 mM Tris-sulfate buffer (pH 7.5), and all further steps were carried out at 0 to 4°C. Cells were washed twice with this buffer before they were resuspended in 10 mM Tris-sulfate buffer containing 1 mM EDTA and 1 mM -mercaptoethanol (TEM buffer) or TEM buffer containing 3 mM NaN₃ (TEMA buffer). After sonication, a crude extract was obtained by centrifugation (200,000 × g, 60 min).

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Construction of HheC mutants. Site-directed mutagenesis was done by using the Quickchange site-directed mutagenesis kit of Stratagene (La Jolla, Calif.) with pGEFHheC as a template. Ser132 was mutated to Cys with the primer set PCODS132C (5'-GGACATATTATCTTTATTACCTGTGCAACGCCCTTCGGGCCTTGG-3') and PNONS132C (5'-CCAAGGCCCGAAGGGCGTTGCACAGGTAATAAAGATAATATGTCC-3') (codon of substituted residue shown in boldface, substituted nucleotides shown in italics). Ser132 was mutated to Ala with the primer set PCODS132A (5'-GGACATATTATCTTTATTACCGCTGCAACGCCCTTCGGGCCTTGG-3') and PNONS132A (5'-CCAAGGCCCGAAGGGCGTTGCAGCGGTAATAAAGATAATATGTCC-3'). Tyr145 was mutated to Phe with the primer set PCODY145F (5'-CCTTGGAAGGAACTTTCTACCTTCACGTCAGCCCGAGCAGGTGC-3') and PNONY145F (5'-GCACCTGCTCGGGCTGACGTGAAGGTAGAAAGTTCCTTCCAAGG-3'). Arg149 was mutated to Lys with the primer set PCODR149K (5'-CCTACACGTCAGCCAAAGCAGGTGCATGCACCTTGGC-3') and PNONR149K (5'-GCCAAGGTGCATGCACCTGCTTTGGCTGACGTGTAGG-3'). Arg149 was mutated to Gln with the primer set PCODR149Q (5'-CCTACACGTCAGCCCAGGCAGGTGCATGCACCTTGGC-3') and PNONR149Q (5'-GCCAAGGTGCATGCACCTGCCTGGGCTGACGTGTAGG-3'). Arg149 was mutated to Glu with the primer set PCODR149E (5'-CCTACACGTCAGCCGAAGCAGGTGCATGCACCTTGGC-3') and PNONR149E (5'-GCCAAGGTGCATGCACCTGCTTCGGCTGACGTGTAGG-3').

CD spectroscopy. Circular dichroism (CD) spectra were recorded with an AVIV 62A DS spectrometer. The far-UV spectra of both the wild type and the HheC variants were recorded at 25°C from 190 to 250 nm with a 0.1-cm cuvette containing 0.1 mg of halohydrin dehalogenase (5 mM potassium phosphate [pH 7.5]) per ml.

Enzyme assays. Halohydrin dehalogenase activities were assayed at 30°C in 50 mM Tris-sulfate buffer (pH 8.0) containing 5 mM substrate by monitoring halide liberation or epoxide formation or with a colorimetric assay using the chromogenic substrate p-nitro-2-bromo-1-phenylethanol. Protein concentration and halide liberation were determined as described before (30). Halohydrins and epoxides were analyzed by gas chromatography. Samples (1.5 ml) were extracted with 1.5 ml of diethyl ether containing 0.05 mM 1-chlorohexane, 1-bromohexane, or mesitylene as an internal standard. Extracts were analyzed by split injection of 2 or 4 µl on an HP5 column (model HP 19091J-413; Hewlett-Packard) with helium as a carrier gas. Separation of enantiomers of chiral compounds was carried out with chiral gas chromatography as described before (16).

Nucleotide sequence accession numbers. The nucleotide sequences described in this article have been deposited in the EMBL/DDBJ/GenBank database under the following accession numbers: HheC, AF397296; HheA_AD2, AF397297; HheB_GP1, AY044094.

	RESULTS

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Cloning and sequence analysis of the halohydrin dehalogenase gene from Agrobacterium radiobacter strain AD1. The gram-negative bacterium A. radiobacter strain AD1 was isolated for its capability to use chloropropanols and epichlorohydrin as growth substrates (30). The organism utilizes (R)-2,3-dichloro-1-propanol, whereas the (S)-enantiomer is not degraded (4). This is caused by the high enantioselectivity of the first enzyme in the degradation pathway, the halohydrin dehalogenase (16). To clone the gene encoding the halohydrin dehalogenase, a gene bank of strain AD1 was constructed in E. coli with the cosmid vector pLAFR3. Three active clones were identified when 1,000 cosmid clones were screened for dehalogenase activity with 1,3-dichloro-2-propanol. Sequence analysis of one of the clones, pAD1-9B2, showed the presence of a complete open reading frame of 765 nucleotides, designated hheC. The first 104 bp were identical to a fragment of a putative halohydrin dehalogenase gene that was located on a genomic DNA segment of A. radiobacter strain CFZ11 that carried the epoxide hydrolase gene (echA) (23). The deduced protein, HheC, has a predicted molecular mass of 27,954 Da and is highly similar to HalB of Agrobacterium tumefaciens (Table 1), for which no biochemical characterization has been published. The hheC gene was amplified by PCR, and the start codon was fused into the NcoI site of pGEF+. The resulting expression vector was designated pGEFHheC.

Cloning and sequence analysis of the halohydrin dehalogenase gene of Arthrobacter sp. strain AD2. Previously, Van den Wijngaard et al. (31) purified the halohydrin dehalogenase of the 3-chloro-1,2-propanediol-degrading Arthrobacter sp. strain AD2. Out of the 34 N-terminal residues, 32 were identical to the halohydrin dehalogenase HheA of Corynebacterium sp. strain N-1074 (36), which suggests that the halohydrin dehalogenase genes of both strains are highly similar. Therefore, primers designed for the hheA gene were used to amplify the halohydrin dehalogenase gene of strain AD2. Three independent clones were sequenced and found to have identical DNA sequences. The gene encodes a 244-amino-acid protein that was designated HheA_AD2, since it was identical to HheA except for 7 amino acid substitutions (Table 1). The gene was fused into the start codon of pGEF+, and the resulting plasmid was designated pGEFHheA_AD2.

Cloning and sequence analysis of the halohydrin dehalogenase gene of Mycobacterium sp. strain GP1. At least two dehalogenases are produced by Mycobacterium sp. strain GP1 when 1,2-dibromoethane serves as a growth substrate (22). A haloalkane dehalogenase, encoded by dhaA_f, catalyzes the hydrolytic dehalogenation of 1,2-dibromoethane to 2-bromoethanol, which is subsequently dehalogenated by a halohydrin dehalogenase to epoxyethane. No halohydrin dehalogenase-producing clones were identified when a gene library of strain GP1 in E. coli was screened for dehalogenase activity with 1,3-dichloro-2-propanol, suggesting that transcription or translation signals were not recognized in E. coli (22). However, when cosmid pGP1-4B5, which carries dhaA_f, was transferred to Pseudomonas sp. strain GJ1 or Burkholderia cepacia G4, the resulting transconjugants rapidly dehalogenated 1,3-dichloro-2-propanol, indicating that active halohydrin dehalogenase was produced. Hence, pGP1-4B5 also harbors the gene encoding the halohydrin dehalogenase of strain GP1. Sequencing showed that the halohydrin dehalogenase gene is located 2,637 bp downstream of dhaA_f and encodes a 235-amino-acid protein. The gene was identical to dehalogenase gene hheB of Corynebacterium sp. strain N-1074, except for 4 nucleotide substitutions that result in 4 amino acid substitutions in the encoded protein (Table 1). As in strain N-1074, a duplication of a 27-nucleotide region (36) has resulted in a duplicate set of AGGA ribosome binding sites, each located 12 nucleotides upstream of an ATG start codon. Hence, two polypeptides can be produced, which differ in the presence or absence of a MANGRKRE amino acid sequence extension at the N terminus, by translation starting from the first or the second start codon, respectively. Previously, Yu et al. (36) have shown that HheB is active as a tetramer and that all possible combinations of the two slightly different subunits occur. However, this subunit composition had little effect on substrate specificity, since enzyme variants exclusively composed of either the long or the short protein were kinetically indistinguishable. The hheB_GP1 gene was amplified by PCR, and the second possible start codon was fused into the NcoI site of pGEF+ to yield the expression vector pGEFHheB_GP1. In this way, a homotetrameric protein consisting only of the shorter 245-amino-acid polypeptide was produced.

Substrate range of halohydrin dehalogenases. All proteins were expressed in a soluble and active form up to 15 to 25% of the total cellular protein content of E. coli BL21(DE3) as judged by SDS-PAGE. Further experiments focused on HheC, because it is enantioselective with various valuable halohydrins (16). Moreover, its sequence is very different from that of halohydrin dehalogenases that have previously been characterized. HheC was purified by anion-exchange chromatography followed by hydrophobic interaction chromatography as described in Materials and Methods. Purified HheC displayed optimal activity at around pH 8.0 to 9.0, and the temperature optimum for activity was 50°C.

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Sequence similarities of halohydrin dehalogenases with SDRs. The pairwise sequence identities of the three halohydrin dehalogenases described in this paper are between 24.2 and 32.4% (Table 1). Interestingly, similarity searches with the amino acid sequences of HheA_AD2, HheB_GP1, and HheC in various protein and DNA databases showed that these enzymes are similar to proteins belonging to the family of SDR enzymes. Sequence similarities of up to 25.5% were observed with well-characterized SDR family members (Table 1). The SDR family constitutes a large number of proteins that catalyze oxidation or reduction reactions with NAD(H) or NADP(H) as a cofactor (13). They are active as dimers or tetramers, where each monomer consists of approximately 250 residues. Halohydrin dehalogenases consist of subunits with similar sizes, and HheA (17) and HheB (20) also appear to be active as tetramers.

View larger version (91K): [in this window] [in a new window]   FIG. 1.   Sequence alignment of halohydrin dehalogenases with SDRs with known three-dimensional structures. The sequences were aligned by using the multiple alignment program ClustalW and are shown in decreasing sequence similarity to HheC. Amino acids that are identical in six or more sequences are depicted below the sequence. Positions at which six or more similar residues occur are indicated by an asterisk. The position of the G/A-G/A-X-X-G/A-X-G/A fingerprint typical of the Rossman fold in the SDR family is indicated by $ on top of the alignment. The proposed active site residues in halohydrin dehalogenases are indicated by #. The structural elements identified in three-dimensional structures of SDR family members are underlined. The first line below the sequence alignment shows the secondary structure elements and the nomenclature of 7-hydroxysteroid dehydrogenase (29). -Strands are indicated as double broken lines, and -helices are indicated as single broken lines. The second, third, and fourth lines show the predicted secondary structure elements for HheC, HheAAD2, and HheBGP1, respectively. The nomenclature of the sequences is explained in Table 1.

View larger version (39K): [in this window] [in a new window]   FIG. 2.   Proposed tertiary structure of halohydrin dehalogenase (HheC) of A. radiobacter AD1. A ribbon representation of residues Lys52 to Ile246 of HheC is shown. The active site residues that are critical for catalysis are indicated. Due to low sequence homology, the N-terminal fraction of HheC could not be predicted by using SDR proteins as a template.

Catalytic mechanism of halohydrin dehalogenases. The strict conservation of Ser132, Tyr145, and Arg149 (HheC numbering) in halohydrin dehalogenases suggests a role of these residues similar to that of their counterparts in members of the SDR protein family. Studies of various SDR family members have shown that the active site residues play a role in catalysis, as depicted in Fig. 3A (3, 13). In this mechanism, the tyrosine acts as a catalytic base to extract a proton from the substrate, and simultaneously NAD(P)⁺ accepts a hydride from the carbon atom. Throughout the catalytic cycle, the conserved serine is hydrogen bonded with the substrate oxygen, thus facilitating proper positioning of the substrate and stabilization of the reaction intermediate (29). The conserved Lys149 may be involved in lowering the pK_a of the tyrosine as well as positioning of the ribose moiety of the cofactor via interaction with its 2'- and 3'-hydroxyl groups (29).

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Proposed reaction mechanism of halohydrin dehalogenase. (A) Reaction mechanism and active site residues of SDRs exemplified by 7-hydroxysteroid dehydrogenase of E. coli (adapted from references 13 and 29). Interactions of the conserved lysine residue with the ribose moiety of the cofactor are not included. (B) Proposed reaction mechanism and active site residues of halohydrin dehalogenase exemplified by HheC of A. radiobacter (see text for explanation).

	DISCUSSION

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Three genes encoding different halohydrin dehalogenases were cloned, sequenced, and overexpressed in E. coli. The enzymes have significant sequence similarity to proteins of the SDR family, and several residues that are highly conserved in SDR enzymes are also present in halohydrin dehalogenases. The typical catalytic triad (Ser-Tyr-Lys) observed in SDR enzymes (11, 13, 29) was partially conserved in halohydrin dehalogenases. In the halohydrin dehalogenases, an arginine is observed at the position at which a lysine is found in the SDR family. However, replacement of the catalytic lysine by an arginine is not unique for halohydrin dehalogenases, since it has also occasionally been observed in other members of the SDR family (e.g., in sorbitol-6-phosphate 2-dehydrogenase of Streptococcus mutans [5] and 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus [34]). The observed sequence similarities between halohydrin dehalogenases and proteins of the SDR family strongly point to a common evolutionary origin. Furthermore, kinetic analysis of several HheC mutants revealed that the conserved Ser-Tyr-Arg triad in halohydrin dehalogenases is crucial for efficient catalysis. These results indicate that the dehalogenation reaction catalyzed by halohydrin dehalogenases also shares interesting similarities with the redox reaction that is catalyzed by members of the SDR family of proteins (Fig. 3). These data also suggest that the dehalogenation reaction catalyzed by halohydrin dehalogenases is fundamentally different from that of hydrolytic dehalogenases, since it does not involve a covalent intermediate.

Including the three new sequences presented in this paper, six complete sequences of halohydrin dehalogenases are now available in the databases. Based on sequence identities, they can be divided into three distinct groups that share 23.9 to 32.4% sequence identity (Table 1). Each group is represented by two sequences, but there are some biochemical and immunological data that allow a preliminary assignment of enzymes of which no complete sequence is known. Group A halohydrin dehalogenases consist of the closely related halohydrin dehalogenases HheA and HheA_AD2. The dehalogenase of the unidentified chloropropanol-utilizing strain AD3 and the DehC produced by Arthrobacter erithrii H10a may also belong to this group (2, 31). Group B enzymes are constituted of HheB and HheB_GP1. The DehA of A. erithrii H10a probably also belongs to this group, since sequencing of peptides of a tryptic digest showed 100% homology to the sequence of HheB (2). Finally, group C halohydrin dehalogenases consist of HheC and halohydrin dehalogenase B of Agrobacterium tumefaciens. A recently isolated halohydrin dehalogenase from an Agrobacterium strain probably also belongs to this group, because it exhibits a similar substrate specificity (9).

The halohydrin dehalogenases HheA_AD2, HheB_GP1, and HheC were active with C2, C3, and aromatic halohydrins and exhibit some remarkable differences in substrate range. The enantioselectivity of halohydrin dehalogenases can be useful for the preparation of optically active compounds. Previously, the epoxide hydrolase of Agrobacterium radiobacter sp. strain AD1 has been cloned, overexpressed, and used for the preparation of optically pure epoxides (15, 23). HheC is the second enzyme in the degradation pathway of chloropropanols in this organism that exhibits high enantioselectivity to chiral substrates. Some promising examples of the application of HheC (16) and other halohydrin dehalogenases for the preparation of optically active aliphatic or aromatic epoxides or halohydrins have been described (1, 14). Interestingly, HheB_GP1 exhibits the opposite enantioselectivity for 2-chloro-1-phenylethanol. Thus, several members of the SDR family and the halohydrin dehalogenases display a high enantioselectivity to many chiral substrates. The stereoselectivity may be determined by only a small number of residues that constitute the substrate binding site (18). These residues are located in the substrate binding loop or in the loops linking -strand D with -helix E and -strand E with -helix F. In two highly identical tropinone reductases (Table 1 and Fig. 1) that have opposite enantioselectivities, stereoselectivity is determined by only 5 amino acid residues (19).

The present study provides insight in the structure and catalytic mechanism of halohydrin dehalogenases, but some important mechanistic questions remain. The reverse reaction, the halogenation of epoxides, is also efficiently catalyzed by these enzymes (1, 31). This probably requires the activation and proper positioning of the halide ion that acts as a nucleophile. From sequence alignments, we were not able to identify a halide-binding site, but it may be formed by residues located at the position at which the cofactor is bound in the SDR family. Tryptophan or arginine residues are involved in halide binding in hydrolytic dehalogenases. Three of the four tryptophan residues of HheC are located in the part of the protein that is predicted in the structure model in Fig. 3. Comparisons with structures of proteins belonging to the SDR family suggest these three residues could interact with the substrate. Future experiments will aim at identifying a halide binding site and elucidating the kinetic properties.