Eur. J. Biochem. 202,1217-1222 (1991)

0FEBS 1991 Characterization of the epoxide hydrolase from an epichlorohydrin-degrading Pseudomonas sp. Mariken H. J. JACOBS, Arjan J. VAN DEN WIJNGAARD, Marjan PENTENGA and Dick B. JANSSEN Department of Biochemistry, Groningen Biotechnology Center, University of Groningen, The Netherlands (Received June 24/September 10, 1991) - EJB 91 0821

An epoxide hydrolase was purified to homogeneity from the epichlorohydrin-utilizing bacterium Pseudomonas sp. strain AD1. The enzyme was found to be a monomeric protein with a molecular mass of 35 kDa. With epichlorohydrin as the substrate, the enzyme followed Michaelis-Menten kinetics with a K , value of 0.3 mM and a Vmaxof 34 pmol . min-' . mg protein-'. The epoxide hydrolase catalyzed the hydrolysis of several epoxides, including epichlorohydrin, epibromohydrin, epoxyoctane and styrene epoxide. With all chiral compounds tested, both stereoisomers were converted. Amino acid sequencing of cyanogen bromide-generated peptides did not yield sequences with similarities to other known proteins.

The biodegradation of halogenated aliphatic epoxides is of considerable interest since these compounds are released into the environment from industrial sources or may be formed during the biotransformation of other synthetic chemicals. Epichlorohydrin (3-chloro-1,2-epoxypropane), which is the most important chlorinated epoxide, is a widely used industrial chemical and is mutagenic for prokaryotes and eukaryotes, as well as carcinogenic for rats [ l , 21. Epibromohydrin and epichlorohydrin may be formed as intermediates during the microbial degradation of halogenated propanols [3], which are used as nematocides. Furthermore, chlorinated ethylenes have been found to be converted to reactive epoxides during oxidative transformation mediated by methane monooxygenases [4, 51. Recently, we described bacterial cultures that are capable of metabolizing epichlorohydrin and chloropropanols [3]. Pseudomonas sp. strain AD1 is able to grow on 1,3-dichloro2-propanol and epichlorohydrin as sole carbon source [3]. Dehalogenation of 1,3-dichloropropanol was found to yield epichlorohydrin, which was hydrolyzed by the action of an epoxide hydrolase to 3-chloro-l,2-propanediol.The latter compound was dehalogenated to glycidol which possibly could be converted to glycerol [3]. Other epoxide-metabolizing activities have been described in mammals and fungi, as well as in bacteria. An epoxide hydrolase converting epoxides to diols has been found in mammalian tissues [6-101 and in fungi [ll]. In higher eukaryotes, epoxide hydrolases are involved in detoxification of xenobiotics. As an alternative, epoxides in mammalian ~~

Correspondence to D. B. Janssen, Department of Biochemistry, Croningen Biotechnology Center, University of Groningen, Nijenhorgh 16, NL-9747 AG Groningen, The Netherlands Abbreviation. GC/MS, gas chromatography/mass spectrometry. Enzymes. Epoxide hydrolase (EC 3.3.2.3); epoxysuccinate hydrolase (EC 3.3.2.4); haloalcohol dehalogenase (EC 4.5.-.-); haloalkane dehalogenase (EC 3.8.1.1). Note. The novel amino acid sequence data published here have been submittcd to the EMBL sequence data hank.

tissue may conjugate to glutathione by the action of a glutathione S-transferase [6]. Only a few other bacterial cultures have been reported to be capable of degrading epoxides. Ethylene oxide conversion was found in cell-free extracts of Mycobacterium E20 [12]. This enzyme system required NAD', CoA and an unknown cofactor. Propylene oxide hydrolysis, without the need for a cofactor, was found in Nocardia A60 [13]. Epoxides produced from unsaturated fatty acids may also be hydrolyzed enzymatically [14]. In all these cases, nothing is known about the biochemical properties of the enzyme involved. Only the purification of an epoxide hydrolase involved in the conversion of trans-2,3-epoxysuccinate in Pseudomonas putida has been described [15]. More insight into the characteristics of bacterial epoxidecleaving enzymes is desirable since epoxides may be formed as intermediates during the conversion of various xenobiotic and natural compounds, including alkenes [16, 171, chlorinated alkenes [4, 51 and haloalcohols [3, 18, 191. Epoxides are reactive compounds that could cause toxicity if they are not rapidly further converted [4, 201. In this paper, we report the purification and characterization of the epoxide hydrolase from the epichlorohydrin-utilizing bacterium Pseudomonas sp. strain AD1.

MATERIALS AND METHODS Organism, growth conditions and preparation of extracts Pseudomonas sp. strain AD1 [3] was used in all experiments. The organism was grown in the mineral medium described before 13,211, supplemented with 10 mg/l yeast extract (medium A). Shaking-flask cultures were carried out at 30°C in 1-1 serum flasks containing 150 ml medium. Sterile substrates were added up to 5 mM. Cultures were harvested after 3 days and crude extract was prepared by sonication of cells suspended in 10 mM Tris/sulphate, pH 7.5, containing 1 mM 2mercaptoethanol and 1 mM EDTA (buffer A). Unbroken cells

1218 and debris were removed by centrifugation and the supernatant was used for determination of epoxide hydrolase activities. Large-scale cultivations were carried out in a 10-1 Braun Biostat E fermentor (Braun AG, Melsungen, FRG). Cells were grown in medium A at 30°C and 40% oxygen saturation. Epichlorohydrin was added discontinuously in 2.5-ml portions. The frequency of addition was increased from once every 16 h to once every 30 min, as growth proceeded. The pH was kept at 7.0 by automatic titration with 10% NH40H. Cultures were harvested at a density of 2.3 mg dry cells/ml by continuous centrifugation (Sharples TIE'). After washing the cells, a crude extract was obtained in buffer A by ultrasonic disruption and centrifugation. Preparation of crude extract and all further manipulations with epoxide hydrolase preparations were carried out at 4 "C and in buffers containing 1 mM EDTA and 1 mM 2-mercaptoethanol to prevent inactivation of the enzyme.

Purification of epoxide hydrolase The extract was diluted to a protein concentration of 10 mg/ml and fractionated by stepwise addition of solid ammonium sulfate to 35%, 40%, 45%, 50% and 80% saturation. The precipitate containing the highest activity (40 -45% precipitate) was dissolved in 125 ml buffer A and dialyzed overnight against the same buffer. The dialyzed enzyme solution was applied to a DEAEcellulose column (2.5 cm x 30 cm), previously equilibrated with buffer A. The column was washed with 100 ml buffer A, and elution was carried out with a 0- 1-M linear gradient of ammonium sulfate in buffer A (total volume, 800 ml; flow rate, 30 ml/h; fraction volume, 7.5 ml). The enzyme eluted at an ammonium sulfate concentration of 0.08 -0.18 M. Active fractions were pooled and dialyzed overnight against 5 mM potassium phosphate buffer (pH 6.8). The dialysate was applied to a hydroxylapatite column (2 cm x 30 cm), previously equilibrated with 5 mM phosphate buffer, pH 6.8. The enzyme was eluted with a linear gradient of 5- 100 mM potassium phosphate (pH 6.8; total volume, 800 ml; flow rate, 30 ml/h; fraction volume, 9.8 ml). Active fractions eluted at 18 -25 mM phosphate. These were pooled and concentrated by ammonium sulfate precipitation (80% saturation). The precipitate was dissolved in 2 ml 50 mM potassium phosphate buffer, pH 7.5, containing 150 mM NaCI. 2 0 0 ~ 1concentrated enzyme solution was applied to a Superose 12 column (1 cm x 30 cm), equilibrated with SO mM phosphate buffer, pH 7.5, containing 150 mM NaCl, in a Pharmacia FPLC system (flow rate, 0.3 ml/min; fraction volume, 0.6 ml). This step was repeated 10 times and the active fractions were pooled. The resulting preparation was filter sterilized and stored at 4 "C for further analysis.

200 'C. Activities were calculated from the difference between the rates of epichlorohydrin disappearance in the presence and absence of enzyme. For rapid assays during the purification of the epoxide hydrolase, a coupled assay was used in which the conversion of epibromohydrin to 3-bromo-1,Zpropanediol was followed by measuring bromide liberation in the presence of excess 3bromo-l,2-propanediol dehalogenase. Incubations contained 3 ml 5 mM epibromohydrin in 50 mM Tris/sulfate buffer, pH 7.5, 100 pl (300 m u ) purified haloalcohol dehalogenase from Arthrobacter AD2 [19], andup to 30 mU epoxide hydrolase. Assays were carried out at 30"C, and bromide production (0 - 1 mM) was determined spectrophotometrically with mercuric thiocyanate and ferric ammonium sulfate [22]. Spectrophotometric incubations for the determination of epoxide hydrolase activities were carried out at 30 "C in 1-cm cuvettes containing 950 p1 SO mM phosphate buffer, pH 7.5, 50 pl purified epoxidase and 0.2-20 pl substrate in ethanol to a final concentration of 0.1 mM. The decrease in absorbance was followed as described by Wixtrom and Hammock [23]. This assay was used for cis-stilbene oxide, transstilbene oxide, 1 ,2-epoxy-l-(2-quinolyl)pentaneand 1,2-epoxy-I -(4-nitrophenyl)pentane. Incubations with ['80]Hz0 were carried out in I-ml vials containing 400 pl [180]Hz0,4 pl 2 M Tris/sulphate, pH 8.0, 0.2 pl epichlorohydrin and 100 p1 purified epoxidase. After a 2.5-h incubation at 30"C, 1-pl samples were analyzed by gas chromatography/mass spectrometry (GC/MS). GC/MS was carried out as described earlier [3], using a 25 m x 0.22 mm CP-Wax52-CB column (Chrompack) at 100°C for separation. Aqueous samples containing 3-chloro1,2-propanediol were separated on a CP-Si15-CB column. 1 U enzyme activity is defined as the amount of enzyme catalyzing the degradation of 1 pmol epichlorohydrin/min. Protein concentrations were determined with Coomassie brilliant blue using bovine serum albumin as the standard.

Polyacrylamide gel electrophoresis SDSjPAGE was conducted on 1.S-mm-thick slab gels consisting of 12.5% acrylamide resolving gels and 4% acrylamide stacking gels. Staining was carried out with Coomassie brilliant blue. For localization of the epoxide hydrolase activity on polyacrylamide gels, electrophoresis was carried out as described previously [24]. One part of the gel was stained with Coomassie brilliant blue and the other part was sliced into 19 equal pieces. The slices were assayed for activity using the coupled assay with the dehalogenase from Arthrobacter sp. strain AD2, as described above. Isoelectric focussing on 6.5% polyacrylamide gels containing 2 % ampholytes was carried out by the method of O'Farrell [25] with carrier ampholytes in the pH range 3.5 - 10.

Assays

Epoxide hydrolase activities were determinated by following the degradation of epichlorohydrin using gas chromatography. Substrate ( 5 mM in 50 mM Tris/sulfate, pH 9.0, containing 1 mM EDTA) was incubated at 30°C with a suitable amount of enzyme. At different time points within 21) min, samples were extracted with diethyl ether that contained trichloroethylene as an internal standard, and analyzed by GC using a column (0.2 mm x 25 m) of CP-Sil52-CB (Chrompack) and a flame-ionization detector. The column was kept at 70°C for 3 min followed by a temperature increase of 10'CC/min to

Characterizationof the epoxide hydrolase The molecular mass of denatured and native protein was estimated, respectively, by SDSjPAGE and by gel filtration on a FPLC Superose 12 column. Marker proteins with SDS/ PAGE were ovotransferrin (78 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa): carbonic anhydrase (30 kDa), myoglobin (17 kDa) and cytochrome c (12.3 kDa). Molecular mass standards for gel filtration were alcohol dehydrogenase (1SO kDa), bovine serum albumin, ovalbumin, trvDsin - - inhibitor (22 kDa) and cytochronie c.

1219 Table 1. Purification of the epoxide hydrolase. The enzyme was purified as described under Materials and Methods. Epoxide hydrolase and protein were determined after each step.

Step

Crude cxtract Ammonium sulfate DEAE-cellulose Hydroxylapatite Superose 12

Total protein

Total activity

activity

mg 10851 3 638 551 41 21

U 3906 3020 1074 852 864

U/mg protein 0.36 0.83 1.95 20.8 32.0

The amino acid composition of the enzyme was determined after hydrolysis of the purified and dialyzed enzyme with 6 M HCI at 110°C for 24 h, using a Kontron amino acid analyzer. Tryptophan content was determined after 96 h of hydrolysis in the presence of mercaptoethanesulfonic acid [26]. Cleavage of protein (2 mg) with cyanogen bromide was performed as described by Vereijken et al. [27]. The cleaved material was lyophilized, dissolved in 0.1YO trifluoroacetic acid and separated by HPLC (Waters Assoc.) on a Nucleosil10 Cl column using a gradient of 0 - 67% acetonitril in 0.1% trifluoroacetic acid for elution. Elution was monitored at 214 nm and peak fractions were collected and lyophilized. The N-terminal sequences of the protein and of CNBrgenerated peptides were sequenced by automated Edman degradation and phenylthiohydantoin derivatization in a gasphase protein sequencer (Applied Biosystems, model 477A) by Eurosequence BV, Groningen.

Specific

Purification factor

Yield

-fold

%

1 2 5 58 89

100 77 27 22 22

1 2 3 4

5

Chemicab

All organic compounds used were obtained from commercial sources (Janssen Chimica or Aldrich) and were at least 95% Pure, as determined by gas ChromatOgraPhY. [1801Hz0 was obtained from MSD Isotopes. cis-Stilbene oxide, transstilbene oxide, 1,2-epoxy-1-(2-quinolyl)pentaneand 1,2epoxy-l-(4-nitrophenyl)pentanewere a gift from Dr B. Hammock, University of California at Davis.

RESULTS Regulation of epoxide hydrolase formation Epoxidase levels in crude extracts from cells of Pseudomonas sp. strain AD1 grown with glucose and epichlorohydrin were, respectively, 0.10 U . ml-' . mg protein-' and 0.25 U ml-' mg protein-'. This suggests that the epoxide hydrolase is constitutively produced, and that the prodyction of the epoxide hydrolase could be elevated further by induction with epichlorohydrin.

Fig. 1. Purification of the epoxide hydrolase from Pseudomonas sp. strain AD1. Samples were taken at different stages of epoxide hydrolase purification (Table 1 ) and subjected to SDS/PAGE. Lanes: 1, crude extract; 2, 40 -45% saturated ammonium sulfate precipitate; 3, pooled fractions after the DEAE-cellulose step: 4, pooled fractions after the hydroxylapatite step; 5, pooled fractions after FPLC Superose 12 chromatography.

used during the purification and storage, since this improved the stability of the enzyme. SDSjPAGE revealed that the epoxide hydrolase isolated was essentially pure, with only very faint bands of contaminating protein being visible (Fig. 1). The enzyme was clearly visible in the crude cell extracts as one of the most prominent protein bands. Activity determinations of gel slices obtained after electrophoresis of the enzyme on a non-denaturing polyacrylamide gel revealed that the stained protein band indeed represented epoxide hydrolase (not shown).

Enzyme purification The epoxide hydrolase was purified from cells of strain AD1 grown under fed-batch conditions with epichlorohydrin as carbon source. 27 mg enzyme was obtained from 25 g dry cells. The enzyme was purified. 89-fold with an overall yield of 22% (Table l), suggesting that the hydrolase represents about 1Yoof the total cellular protein. The hydrolase could be stored at 4°C fo 5 months without significant loss of activity. EDTA and 2-mercaptoethanol were included in all buffers

Molecular mass The native epoxide hydrolase showed a molecular mass of 35 kDa, as determined by FPLC on a Superose 12 column. The enzyme had exactly the same elution volume as the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The molecular mass of this enzyme is 35 143 Da, as found by DNA sequencing [28]. The molecular mass of the denatured protein as determined by SDSjPAGE also yielded a value of 35 kDa. It

1220

Table 2. Substrate specificity of the enzyme. Activities were determined by following substrate disappearance by gas chromatography. The rates of degradation of the different substrates are expressed as percentages of the degradation rate observed with epichlorohydrin. Substrate

Relative activity

06 3 00

Epichlorohydrin Epibromohydrin Propylene oxide Glycidol Epoxyoctane Styrene oxide Cyclohexene oxide

0 20

99 20

Characterization of the epoxide hydrolase from an epichlorohydrin-degrading Pseudomonas sp.

An epoxide hydrolase was purified to homogeneity from the epichlorohydrin-utilizing bacterium Pseudomonas sp. strain AD1. The enzyme was found to be a...
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