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. 1999 Dec;65(12):5459–5463. doi: 10.1128/aem.65.12.5459-5463.1999

Isolation and Characterization of the Epoxide Hydrolase-Encoding Gene from Xanthophyllomyces dendrorhous

Hans Visser 1,*, Jan A M de Bont 1, Jan C Verdoes 1
PMCID: PMC91744  PMID: 10584004

Abstract

The epoxide hydrolase (EH)-encoding gene (EPH1) from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the α/β hydrolase fold family. The EPH1 gene was not essential for growth of X. dendrorhous in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in Escherichia coli. A sixfold increase in specific activity was observed when we used resting cells rather than X. dendrorhous. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the X. dendrorhous EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system.

Epoxide hydrolases (EHs) catalyze hydrolysis of epoxides to the corresponding diols. These enzymes have potential industrial uses in fine chemistry, as both enantiomerically pure epoxides and 1,2-diols are central building blocks in the asymmetric synthesis of biologically active molecules (25, 40). EHs are found in both prokaryotic and eukaryotic organisms, where they perform different functions. The best-studied mammalian EHs are involved in detoxification of epoxides derived from both xenobiotic compounds and endogenous substrates (2, 22, 30). Plant EHs are believed to be involved in the biosynthesis of cutin (5) and components of plant defense mechanisms (17). Insect EHs might be involved in the regulation of juvenile hormone titers by degrading the hormone in concert with juvenile hormone esterase (8). Microbial EHs are found in bacteria and fungi, where they perform catabolic functions (4, 6).

Two types of mammalian EHs, soluble EH (sEH) and microsomal EH (mEH), are involved in detoxification of noxious epoxides. Several plant (15, 32) and bacterial (18, 28) EHs are similar to sEH, whereas the insect juvenile hormone EH sequence is similar to the sequence of mEH (42). Both types of EHs belong to the α/β hydrolase fold family of enzymes (1, 28), a large group of proteins that are structurally and mechanistically related (24). Hydrolysis of epoxides by these enzymes occurs via an enzyme-ester-substrate intermediate (1, 16, 19). In addition to the α/β hydrolase fold enzymes, a few EHs belong to other classes of enzymes; these EHs include leukotriene A4 hydrolase (10), cholesterol EH (38), and limonene-1,2-epoxide hydrolase (34). EH activity has been found in several fungi, including Rhodotorula glutinis (39), Aspergillus niger (20), and Beauveria sulfurescens (26), but no EH-encoding gene from a eukaryotic microorganism has been described yet.

Their broad substrate ranges, high enantioselectivities, and reaction rates (40) make yeast EHs promising candidates for industrial applications. However, in most cases wild-type yeast EHs do not meet the exact requirements for specific epoxide conversions in industrial processes. Therefore, our ultimate goal is to develop a yeast EH-based biocatalyst with tailor-made substrate specificity and enantioselectivity. Isolation and characterization of a yeast EH-encoding gene, which would result in information about the gene and gene product which is needed for construction of an improved enzyme via genetic engineering, would be a first step to take. The EH activity and the availability of molecular genetic tools (41) prompted us to isolate and characterize the EH-encoding gene of the basidiomycetous yeast Xanthophyllomyces dendrorhous.

MATERIALS AND METHODS

Strains, plasmids, and DNA manipulation.

Strain CBS 6938 of X. dendrorhous, the perfect state of Phaffia rhodozyma (12), was used throughout this study. Escherichia coli XL1-Blue-MRF′ (Stratagene, La Jolla, Calif.) was used for transformation and expression experiments. The vector pGEM-T Easy (Promega Benelux BV, Leiden, The Netherlands) and the prokaryotic expression vector pKK223-3 (Pharmacia Biotech Benelux, Roosendaal, The Netherlands) were used for cloning and expression experiments, respectively, performed with E. coli. To construct pEHKO, a 750-bp EPH1 fragment that corresponded to the C-terminal half of the enzyme and had a unique EcoRV restriction site was amplified by performing a PCR with primers PXD2 and PXD3 and with cDNA as the template. The PCR product was cloned into pGEM-T Easy, and the truncated EPH1 fragment was released as a 776-bp EcoRI fragment from the plasmid. The fragment was purified and ligated into the unique EcoRI restriction site of X. dendrorhous transformation vector pPR1T. The newly constructed vector was designated pEHKO. Plasmid pPR1T was constructed by ligating a 0.3-kb BamHI-HindIII PCR product that corresponded to the glyceraldehyde-3-phosphate dehydrogenase terminator sequence of X. dendrorhous (36) into the corresponding restriction sites of X. dendrorhous transformation vector pPR1 (41). Unless indicated otherwise, standard molecular cloning techniques were used (29). Chromosomal DNA was isolated from sodium dodecyl sulfate-lysed protoplasts of X. dendrorhous and was extracted with phenol-chloroform as described previously (36). Plasmid DNA from E. coli was isolated by using Qiagen columns (Westburg BV, Leusden, The Netherlands). DNA trapped in an agarose gel was isolated by using a QIAEX II gel extraction kit. A nonradioactive digoxygenin DNA labeling and detection kit (Roche Diagnostics Nederland BV, Almere, The Netherlands) was used to label and detect probes in Southern blot or colony hybridization preparations. Construction of the X. dendrorhous genomic and cDNA libraries was described previously (36). Standard nomenclature for yeast genes and proteins was used to name the X. dendrorhous EH gene and protein (33).

Transformation of X. dendrorhous and E. coli by electroporation.

Electrocompetent cells of X. dendrorhous were prepared as described by Wery et al. (41). Electrocompetent cells were mixed with 5 μg of linearized DNA, and the mixture was transferred to a precooled electroporation cuvette (length, 0.2 cm). A 0.8-kV, 1,000-Ω, 25-μF pulse was applied with a gene pulser (Bio-Rad Laboratories BV, Veenendaal, The Netherlands). Then 0.5 ml of YePD medium containing 1% yeast extract, 2% Bacto Peptone (Difco), and 2% glucose was added immediately, and the mixture was transferred to a sterile 1.5-ml Eppendorf tube. After incubation for 2.5 h at 21°C, 100-μl aliquots were spread onto solid YePD medium plates containing 40 μg of G418 per ml. The plates were incubated at 21°C until colonies appeared.

E. coli was cultivated in Luria-Bertani (LB) (29) medium lacking NaCl to an optical density at 600 nm of 0.5. The cells were washed twice in an equal volume of ice-cold demineralized water and once in an equal volume of 10% glycerol in demineralized water. After centrifugation (1,000 × g, 10 min) the cells were resuspended in a 0.05 volume of 10% glycerol in demineralized water. One hundred microliters of cells was mixed with plasmid DNA and transferred to an electroporation cuvette (length, 0.2 cm). A 2.5-kV, 200-Ω, 25-μF pulse was applied, and then 1 ml of SOC medium (29) was added immediately. The transformation mixture was transferred to a 1.5-ml Eppendorf tube and incubated at 37°C for 1 h. Aliquots were spread onto solid LB agar plates containing 50 μg of ampicillin per ml.

PCR.

Standard PCR were carried out with an automated thermal cycler (Perkin-Elmer Nederland, Nieuwerkerk a/d IJssel, The Netherlands). The reaction conditions were as follows: 5 min at 94°C, followed by 30 cycles consisting of 1 min at 94°C, 2 min at 45 or 50°C, and 1 or 2.5 min at 72°C and a final step consisting of 10 min at 72°C. The following oligonucleotide primers were used: PANEH2 (5′-TCNCTBCCYGGHTAYACNTTYTC-3′) (which encodes SLPGYTFS of Aspergillus niger EH), PANEH4A (5′-TCVAGWGCWGCRAARTGDCCDCC-3′) (which encodes the antisense sequence GGHFAALE of A. niger EH), PXD2 (5′-CACTGCCCGGCTATACGTTCTCCTCTGGTCCGCAACG-3′) (which encodes LPGYTFSSGPQR of X. dendrorhous EH), and PXD3 (5′-CAAGAGCAGCAGCAAAGTGTCCGCCTCGAGCATG-3′) (which encodes the antisense sequence HARGGHFAAL of X. dendrorhous EH) (the underlined nucleotides correspond to parts of degenerate oligonucleotides PANEH2 and PANEH4A found in pxdeh1). We also used primer PROKATG (5′-TTTGAATTCATGACGTCTGCGACATTCCTAC-3′) and primer PROKTAA (5′-AGCTCTGCAGTTAAAGCTCGGAATGATAGTTC-3′) (the underlined nucleotides are restriction sites introduced for subcloning purposes).

EH enzyme assay.

X. dendrorhous strains were grown in high-cell-density cultures for 48 h at 21°C in 400 ml of YePD medium. E. coli(pKK223-3) and E. coli(precEph1) were grown overnight in 100 ml of LB medium supplemented with ampicillin (50 μg/ml) and isopropyl-β-d-thiogalactopyranoside (IPTG) (0.84 mM). Cells were harvested by centrifugation (1,000 × g, 10 min) and washed in 50 mM potassium phosphate buffer (pH 7.0). Cell pellets were resuspended in 3 ml of 50 mM potassium phosphate buffer (pH 7.0) to obtain concentrated suspensions of resting cells. One-milliliter portions of these suspensions were mixed with 5 mM racemic substrate by vortexing the preparations in sealed tubes or bottles and incubating them with shaking at 35°C in a water bath. To monitor epoxide degradation, headspace samples were taken over a period of time and were analyzed by gas chromatography by using a Chrompack model CP9000 gas chromatograph equipped with an α- or β-cyclodextrin 120 chiral column (Supelco Inc.). Racemic 1,2-epoxyhexane (Aldrich, Zwijndrecht, The Netherlands) was analyzed by using β-cyclodextrin (detection limit, 50 μM). Racemic 1-methylcyclohexene oxide (MCHO) was analyzed by using α-cyclodextrin (detection limit, 50 μM). To determine the protein content, 1 ml of a diluted sample was added to 0.5 ml of 0.5 N NaOH and incubated at 100°C for 30 min. Subsequently, 100 μl of this preparation was used in the DC protein assay (Bio-Rad).

Nucleotide sequence accession number.

The nucleotide sequence of the EPH1 gene has been deposited in the GenBank database under accession no. AF166258.

RESULTS

Synthesis of a specific X. dendrorhous EH probe by PCR.

Initial enzyme localization studies showed that most EH activity was located in the membrane fraction of a cell extract of X. dendrorhous (results not shown). Therefore, we surmised that X. dendrorhous contains an mEH. We aligned the amino acid sequences of human (31), rabbit (14), rat (27), and Manduca sexta (42) mEHs and identified conserved regions. Several conserved peptide sequences were used to design degenerate oligonucleotides which could be used as primers in a PCR performed with X. dendrorhous chromosomal DNA as the template, but no X. dendrorhous EH fragment was amplified. We then used the conserved portion of the amino acid sequence of A. niger EH (11) to design degenerate oligonucleotides by using X. dendrorhous preferred codons (36). A 1.2-kb fragment was amplified in a PCR performed with oligonucleotides PANEH2 and PANEH4. This fragment was cloned into pGEM-T Easy, which yielded pxdeh1, and the nucleotide sequence of the PCR fragment was determined. A comparison of the deduced amino acid sequence to the sequences in protein databases revealed homology to the C-terminal parts of eukaryotic mEHs (e.g., rat, mouse, and rabbit mEHs). This result indicated that a 1.2-kb fragment of the putative X. dendrorhous EH-encoding gene, designated EPH1, was cloned.

Isolation of the X. dendrorhous EPH1 gene.

The 1.2-kb fragment of pxdeh1 was used to screen an X. dendrorhous cDNA and genomic cosmid library. The EPH1 cDNA contained a 1,236-bp open reading frame which corresponded to a 411-amino-acid polypeptide with a calculated molecular mass of 46,185 Da. The two highest values resulting from a comparison of the total Eph1 amino acid sequence to the SwissProt protein database sequences were the values for pig mEH (32.5% identity) and rabbit mEH (32.2% identity). A comparison of the pxdeh1 nucleotide sequence with cDNA sequences revealed that several introns were present.

Cosmid pEHcos8 hybridized to pxdeh1 in a colony hybridization screening experiment, indicating that this cosmid carries an X. dendrorhous chromosomal DNA fragment containing part or all of the EPH1 gene. The nucleotide sequence of the EPH1 gene was determined by sequencing pEHcos8. Initially, this was done by using specific oligonucleotides designed by using the pxdeh1 sequence, and subsequently it was done by using a primer walking strategy. We sequenced 2,931 bp, including the EPH1 gene, 716 bp upstream of the putative translation initiation codon, and 219 bp downstream of the putative TAA stop codon. The EPH1 open reading frame was interrupted by eight introns, whose sizes varied between 61 and 110 bp. The promoter region contained putative elements, such as a CCAAT box, a TATA box, and a CAP signal. The 3′ nontranslated region contained the sequence TATGTATG…TATGT…TTT, which was similar to 3′ nontranslated regions of several Saccharomyces cerevisiae genes (21, 43).

Inactivation of the EPH1 gene in X. dendrorhous by transformation-mediated gene disruption.

We inactivated the chromosomal EPH1 gene by transformation-mediated gene disruption. We linearized pEHKO (Fig. 1) by digesting it with EcoRV and introduced it into X. dendrorhous CBS6938 by electrotransformation (41). Transformants were selected on the basis of G418 resistance. Both X. dendrorhous CBS6938 and putative EPH1− deletion mutants, which were designated CBS6938(pEHKO), were cultivated for 48 h at 21°C in rich medium (YePD medium) until they reached the stationary growth phase. Apparently, the mutation did not have a lethal effect on X. dendrorhous, and EPH1 was not essential for growth under these laboratory conditions.

FIG. 1.

FIG. 1

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Transformation-mediated inactivation of the EPH1 gene: schematic representation of a single copy and multiple copies of plasmid pEHKO integrated into the EPH1 gene. The first EPH1 exon is represented by a black box with an arrowhead to indicate the EPH1 direction. Plasmid pEHKO was constructed by cloning a 776-bp EcoRI PCR fragment containing the truncated EPH1 gene copy (corresponding to amino acids 138 to 388) in X. dendrorhous transformation vector pPR1T. Prior to electroporation plasmid pEHKO was linearized by digestion with EcoRV.

Both a racemic aliphatic epoxide (1,2-epoxyhexane) and a racemic alicyclic epoxide (MCHO) were used as substrates to study Eph1 activity. X. dendrorhous hydrolyzed 1,2-epoxyhexane with low enantioselectivity at an initial reaction rate of 4.8 nmol/min/mg of protein. MCHO was enantioselectively hydrolyzed at a rate of 0.17 nmol/min/mg of protein by the wild-type strain. No EH activity was observed with either substrate when transformants CBS6938(pEHKO)1 and CBS6938(pEHKO)2 were used.

We probed DNA from the two transformants on Southern blots with the 1.2-kb EcoRI fragment of pxdeh1 (Fig. 2). Digestion of wild-type X. dendrorhous CBS6938 DNA with EcoRI resulted in a large (>10-kb) hybridizing fragment. Digestion of CBS6938(pEHKO) with EcoRI resulted in three fragments. The presence of the approximately 750-bp EcoRI fragment from CBS6938(pEHKO) could be explained by multiple-copy integration of pEHKO into the EPH1 gene (Fig. 1). This explanation was confirmed by the hybridization patterns of genomic DNA digested with EcoRV or EcoRI plus EcoRV. Digestion of CBS6938 DNA with EcoRV resulted in two fragments (705 and 805 bp), whereas digestion of CBS6938(pEHKO) DNA with EcoRV resulted in the expected wild-type pattern plus an additional 5.2-kb fragment, whose size corresponded to the size of the linear form of pEHKO. An additional fragment at approximately 385 bp was also produced, and this fragment corresponded to the equally large EcoRI-EcoRV fragments of pEHKO. A similar pattern was observed when pEHKO was digested with the same endonucleases.

FIG. 2.

FIG. 2

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Transformation-mediated inactivation of the EPH1 gene: autoradiograph resulting from the Southern blot analysis of chromosomal DNA of wild-type CBS6938 and transformants CBS6938(pEHKO)1 and CBS6938(pEHKO)2. Lanes 1, 4, and 7, chromosomal DNA digested with EcoRI; lanes 2, 5, and 8, chromosomal DNA digested with EcoRV; lanes 3, 6, and 9, chromosomal DNA digested with EcoRI and EcoRV; lane 10, plasmid pEHKO DNA digested with EcoRI. The positions of the hybridizing DNA fragments whose sizes corresponded to the size of plasmid pEHKO (5.2 kb) in lanes 5 and 8 and the approximately 750-bp, truncated-EPH1-containing, EcoRI fragment of pEHKO in lanes 4, 7, and 10 are indicated by arrows.

Functional expression of recombinant Eph1 in E. coli.

We expressed the Eph1-encoding cDNA in E. coli. Eph1-encoding cDNA was amplified in a PWO polymerase (Roche Diagnostics Nederland BV)-catalyzed PCR by using forward primer PROKATG and reverse primer PROKTAA with EPH1 cDNA as the template. The 1,255-bp PCR product was digested with EcoRI and PstI and ligated into the corresponding sites of prokaryotic expression vector pKK223-3, which yielded precEph1. E. coli(precEph1) was subjected to the EH enzyme assay. The initial reaction rates of E. coli(precEph1) with 1,2-epoxyhexane and MCHO were 31 and 1 nmol/min/mg of protein, respectively; these values were approximately sixfold higher than the values obtained with wild-type X. dendrorhous. As expected, no EH activity was detected when E. coli(pKK223-3) was used. Unlike 1,2-epoxyhexane, MCHO was selectively degraded by both native Eph1 and recombinant Eph1 (recEph1) (Fig. 3); 1R,2S-MCHO was degraded faster than 1S,2R-MCHO. After 1R,2S-MCHO was completely degraded, the yield of 1S,2R-MCHO was 30%.

FIG. 3.

FIG. 3

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Functional expression of X. dendrorhous EPH1 in E. coli. Hydrolysis of 5 mM racemic MCHO was monitored over a period of time. Symbols: ■, 1S,2R-MCHO (35); ⧫, 1R,2S-MCHO (35).

DISCUSSION

Eph1 from the basidiomycetous yeast X. dendrorhous resembles mammalian mEHs and belongs to the α/β hydrolase fold family of enzymes, which consists of a large number of enzymes, including sEHs and mEHs (1, 24). Several conserved sequence motifs have been found in this family of enzymes, especially in the region of the highly conserved nucleophile-histidine-acid catalytic triad (24). The consensus sequence of the nucleophile motif is sm-x-nu-x-sm-sm, where sm is a small amino acid, x is any amino acid, and nu is the nucleophile. The HGXP motif contains the oxyanion hole of the enzyme (1, 16, 24). The three putative members of the Eph1 catalytic triad are an aspartate residue at position 181, corresponding to the nucleophile motif, a glutamate residue at position 359, which aligns with the catalytic acidic residue, and a histidine residue at position 385, which could be the catalytic histidine (Fig. 4).

FIG. 4.

FIG. 4

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Comparison of the amino acid sequences of Eph1 and mEHs: alignment of the amino acid sequences of X. dendrorhous EH (Eph1) (this study), rabbit mEH (RBmEH) (14) (SwissProt datafile accession no. P04068), and rat mEH (RTmEH) (27) (P07687). The asterisks indicate positions that are perfectly conserved; the apostrophes indicate positions that are well conserved. The residues that form the catalytic triad are indicated by N (nucleophile), A (acidic residue), and H (histidine). The amino acid sequence corresponding to the nucleophile motif is underlined with one line. The amino acid sequence corresponding to the HGXP motif is underlined with two lines.

The predicted Eph1 polypeptide (411 amino acids) of X. dendrorhous is shorter than most mammalian mEHs (455 amino acids) and the juvenile hormone EH (462 amino acids). The differences are mainly found in the N-terminal regions of these enzymes (Fig. 4). mEH is cotranslationally inserted into the endoplasmic reticulum (13, 23) by means of a single membrane anchor segment in the N terminus of the enzyme (9). This segment is missing from Eph1, and its absence may partially explain the different lengths of mammalian mEHs and Eph1. Even though Eph1 lacks a membrane anchor segment, it appears to be membrane associated since most of the EH activity was found in the membrane fraction of an X. dendrorhous cell extract.

Protein engineering, in which both specific and random amino acids were substituted, has been shown to be a powerful tool for improving enzyme characteristics (7). For mEH, it has been shown that replacing the catalytic acidic residue (Glu at position 404) with an aspartate residue greatly increases the Vmax of this enzyme (3). We demonstrated that EPH1 cDNA is expressed in E. coli. This expression system is a powerful tool for analyzing Eph1 mutants (e.g., Glu-359–Asp) created by protein engineering. Furthermore, it is a simple system which can be used to study the substrate range of mutant Eph1. In addition, the Eph1 amino acid sequence information may be helpful in cloning other yeast EH-encoding genes.

Recently, we isolated the EH-encoding gene from the yeast Rhodotorula glutinis by using the X. dendrorhous Eph1 sequence information (37). A comparison of the amino acid sequences and substrate specificities should provide new insights that can be used for protein-engineering experiments.

ACKNOWLEDGMENTS

We thank R. Furstoss (Faculté des Sciences de Luminy, Marseille, France) and M. Arand (Institute of Toxicology, University of Mainz, Mainz, Germany) for providing the A. niger EH amino acid sequence and C. A. G. M. Weijers (Division of Industrial Microbiology, Wageningen Agricultural University, Wageningen, The Netherlands) for providing MCHO.

This work was supported by grant BIOC4-CT95-0005 from the European Commission.

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