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Active Site Engineering of the Epoxide Hydrolase from Agrobacterium radiobacter AD1 to Enhance Aerobic Mineralization of cis-1,2-Dichloroethylene in Cells Expressing an Evolved Toluene ortho-Monooxygenase

2004; Elsevier BV; Volume: 279; Issue: 45 Linguagem: Inglês

10.1074/jbc.m407466200

ISSN

1083-351X

Autores

Lingyun Rui, Li Cao, Wilfred Chen, Kenneth F. Reardon, Thomas K. Wood,

Tópico(s)

Enzyme Catalysis and Immobilization

Resumo

Chlorinated ethenes are the most prevalent ground-water pollutants, and the toxic epoxides generated during their aerobic biodegradation limit the extent of transformation. Hydrolysis of the toxic epoxide by epoxide hydrolases represents the major biological detoxification strategy; however, chlorinated epoxyethanes are not accepted by known bacterial epoxide hydrolases. Here, the epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA), which enables growth on epichlorohydrin, was tuned to accept cis-1,2-dichloroepoxyethane as a substrate by accumulating beneficial mutations from three rounds of saturation mutagenesis at three selected active site residues, Phe-108, Ile-219, and Cys-248 (no beneficial mutations were found at position Ile-111). The EchA F108L/I219L/C248I variant coexpressed with a DNA-shuffled toluene ortho-monooxygenase, which initiates attack on the chlorinated ethene, enhanced the degradation of cis-dichloroethylene (cis-DCE) an infinite extent compared with wild-type EchA at low concentrations (6.8 μm) and up to 10-fold at high concentrations (540 μm). EchA variants with single mutations (F108L, I219F, or C248I) enhanced cis-DCE mineralization 2.5-fold (540 μm), and EchA variants with double mutations, I219L/C248I and F108L/C248I, increased cis-DCE mineralization 4- and 7-fold, respectively (540 μm). For complete degradation of cis-DCE to chloride ions, the apparent Vmax/Km for the Escherichia coli strain expressing recombinant the EchA F108L/I219L/C248I variant was increased over 5-fold as a result of the evolution of EchA. The EchA F108L/I219L/C248I variant also had enhanced activity for 1,2-epoxyhexane (2-fold) and the natural substrate epichlorohydrin (6-fold). Chlorinated ethenes are the most prevalent ground-water pollutants, and the toxic epoxides generated during their aerobic biodegradation limit the extent of transformation. Hydrolysis of the toxic epoxide by epoxide hydrolases represents the major biological detoxification strategy; however, chlorinated epoxyethanes are not accepted by known bacterial epoxide hydrolases. Here, the epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA), which enables growth on epichlorohydrin, was tuned to accept cis-1,2-dichloroepoxyethane as a substrate by accumulating beneficial mutations from three rounds of saturation mutagenesis at three selected active site residues, Phe-108, Ile-219, and Cys-248 (no beneficial mutations were found at position Ile-111). The EchA F108L/I219L/C248I variant coexpressed with a DNA-shuffled toluene ortho-monooxygenase, which initiates attack on the chlorinated ethene, enhanced the degradation of cis-dichloroethylene (cis-DCE) an infinite extent compared with wild-type EchA at low concentrations (6.8 μm) and up to 10-fold at high concentrations (540 μm). EchA variants with single mutations (F108L, I219F, or C248I) enhanced cis-DCE mineralization 2.5-fold (540 μm), and EchA variants with double mutations, I219L/C248I and F108L/C248I, increased cis-DCE mineralization 4- and 7-fold, respectively (540 μm). For complete degradation of cis-DCE to chloride ions, the apparent Vmax/Km for the Escherichia coli strain expressing recombinant the EchA F108L/I219L/C248I variant was increased over 5-fold as a result of the evolution of EchA. The EchA F108L/I219L/C248I variant also had enhanced activity for 1,2-epoxyhexane (2-fold) and the natural substrate epichlorohydrin (6-fold). Epoxide hydrolases (EH) 1The abbreviations used are: EH, epoxide hydrolase; TCE, trichloroethylene; DCE, dichloroethylene; TOM, toluene ortho-monooxygenase; Kan, kanamycin; GC, gas chromatography; AnEH, Aspergillus niger epoxide hydrolase; EchA, epoxide hydrolase from Agrobacterium radiobacter AD1. (EC 3.3.2.3) hydrolyze an epoxide to its corresponding vicinal diol by the addition of a water molecule (1Weijers C.A.G.M. de Bont J.A.M. J. Mol. Catal. B Enzym. 1999; 6: 199-214Crossref Scopus (108) Google Scholar). In mammalian systems, epoxides are frequently found as intermediates in the catabolic pathways of various xenobiotics, including unsaturated aliphatic and aromatic hydrocarbons (2Zou J. Hallberg B.M. Bergfors T. Oesch F. Arand M. Mowbray S.L. Jones T.A. Structure. 2000; 8: 111-122Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 3Fretland A.J. Omiecinski C.J. Chem. Biol. Interact. 2000; 129: 41-59Crossref PubMed Scopus (258) Google Scholar, 4Hernandez O. Bend J.R. Jakoby W.B. Bend J.R. Caldwell J. Metabolic Basis of Detoxification. Academic Press, New York1982: 207-208Google Scholar). These intermediates are potentially harmful as the oxirane moiety of epoxides is electrophilically reactive and can form adducts with various cellular components including DNA, RNA, proteins, and other small molecules (5Archelas A. Furstoss R. Trends Biotechnol. 1998; 16: 108-116Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 6van Hylckama Vlieg J.E.T. Janssen D.B. J. Biotechnol. 2001; 85: 81-102Crossref PubMed Scopus (62) Google Scholar); hence, it is vital for the biological system to detoxify these reactive species. Together with the conjugation reaction catalyzed by glutathione S-transferases, conversion of epoxides by EHs into chemically and toxicologically less active diols constitutes the major mechanism for detoxification in mammalian systems (5Archelas A. Furstoss R. Trends Biotechnol. 1998; 16: 108-116Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Although mammalian EHs have been extensively studied for detoxification, interest in microbial EHs has arisen primarily because of the potential of the enzymes as enantioselective biocatalysts (1Weijers C.A.G.M. de Bont J.A.M. J. Mol. Catal. B Enzym. 1999; 6: 199-214Crossref Scopus (108) Google Scholar, 7Faber K. Mischitz M. Kroutil W. Acta Chem. Scand. 1996; 50: 249-258Crossref Scopus (89) Google Scholar, 8Steinreiber A. Faber K. Curr. Opin. Biotechnol. 2001; 12: 552-558Crossref PubMed Scopus (168) Google Scholar). It is also of interest to investigate the detoxification role of microbial EHs and genetically adapt this universally successful detoxification strategy to the process of aerobic, cometabolic biodegradation of chlorinated ethenes in which toxic epoxides form as the primary intermediates (6van Hylckama Vlieg J.E.T. Janssen D.B. J. Biotechnol. 2001; 85: 81-102Crossref PubMed Scopus (62) Google Scholar). Chlorinated ethenes, such as trichloroethylene (TCE) and cis-1,2-dichloroethylene (cis-DCE), constitute a large group of priority pollutants (126 chemicals from the U.S. Clean Water Act) (9Bradley P.M. Chapelle F.H. Environ. Sci. Technol. 1998; 32: 553-557Crossref Scopus (84) Google Scholar, 10McCarty P.L. Science. 1997; 276: 1521-1522Crossref PubMed Scopus (197) Google Scholar). Because they are toxic, it is critical to remediate these compounds (11Henschler D. Angew. Chem. Int. Ed. Engl. 1994; 33: 1920-1935Crossref Scopus (116) Google Scholar). Whereas reductive dechlorination of chlorinated ethenes under anaerobic conditions has the risk of accumulation of the well known carcinogen, vinyl chloride (11Henschler D. Angew. Chem. Int. Ed. Engl. 1994; 33: 1920-1935Crossref Scopus (116) Google Scholar, 12Bolt H.M. Lair R.J. Filser J.G. Biochem. 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Wackett L.P. J. Bacteriol. 1997; 179: 90-96Crossref PubMed Google Scholar, 16van Hylckama Vlieg J.E.T. de Koning W. Janssen D.B. Appl. Environ. Microbiol. 1996; 62: 3304-3312Crossref PubMed Google Scholar, 17van Hylckama Vlieg J.E.T. de Koning W. Janssen D.B. Appl. Environ. Microbiol. 1997; 63: 4961-4964Crossref PubMed Google Scholar, 18Yeager C.M. Bottomley P.J. Arp D.J. Appl. Environ. Microbiol. 2001; 67: 2107-2115Crossref PubMed Scopus (44) Google Scholar). Recently, a novel glutathione S-transferase from Rhodococcus sp. strain AD45 having activity toward cis-1,2-dichloroepoxyethane (cis-DCE epoxide) was coexpressed with an evolved toluene ortho-monooxygenase (TOM), TOM-Green, in E. coli in our laboratory. It showed significant detoxification of the reactive epoxide intermediates from cis-DCE, trans-1,2-dichloroethylene, and TCE (19Rui L. Kwon Y.M. Reardon K.F. Wood T.K. Environ. Microbiol. 2004; 6: 491-500Crossref PubMed Scopus (33) Google Scholar) (Fig. 1). TOM is a three-component, diiron enzyme encoded by the Burkholderia cepacia G4 genes tomA012345 (20.Shields, M. S., and Francesconi, S. C. (August 6, 1996) U. S. Patent 5,543,317Google Scholar), catalyzing hydroxylation of toluene to form 3-methylcatechol through the intermediate o-cresol (21Shields M.S. Montgomery S.O. Chapman P.J. Cuskey S.M. Pritchard P.H. Appl. Environ. Microbiol. 1989; 55: 1624-1629Crossref PubMed Google Scholar). TOM also oxidizes TCE primarily to Cl– and CO2in vivo (22Luu P.P. Yung C.W. Sun A.K. Wood T.K. Appl. Microbiol. Biotechnol. 1995; 44: 259-264Crossref Scopus (1) Google Scholar, 23Nelson M.J.K. Montgomery S.O. O'Neill E.J. Pritchard P.H. Appl. Environ. Microbiol. 1986; 52: 383-384Crossref PubMed Google Scholar) and aerobically degrades various other chlorinated ethenes (20.Shields, M. S., and Francesconi, S. C. (August 6, 1996) U. S. Patent 5,543,317Google Scholar, 24Shields M.S. Reagin M.J. Gerger R.R. Somerville C. Schaubhut R. Campbell R. Hu-Primmer J. Hinchee R.E. Leeson A. Semprini L. Ong S.K. Bioremediation of Chlorinated and Polycyclic Aromatic Hydrocarbon Compounds. Lewis Publishers, Boca Raton, FL1994: 50-65Google Scholar, 25Shim H. Wood T.K. Biotechnol. Bioeng. 2000; 70: 693-698Crossref PubMed Scopus (33) Google Scholar). TOM-Green originated from the first DNA shuffling of a non-heme monooxygenase and has enhanced activity for both TCE degradation and naphthalene oxidation due to a single amino acid substitution, V106A, in TomA3 (26Canada K.A. Iwashita S. Shim H. Wood T.K. J. Bacteriol. 2002; 184: 344-349Crossref PubMed Scopus (154) Google Scholar). In contrast to glutathione S-transferases, which require glutathione as the cofactor for their enzymatic activity (27Vuilleumier S. J. Bacteriol. 1997; 179: 1431-1441Crossref PubMed Google Scholar), EHs do not require a cofactor (1Weijers C.A.G.M. de Bont J.A.M. J. Mol. Catal. B Enzym. 1999; 6: 199-214Crossref Scopus (108) Google Scholar). Unfortunately, there are no EHs of microbial origin known to have activity toward chlorinated epoxyethanes. Nevertheless, a number of microorganisms contain EHs with various substrate ranges (28Jacobs M.H. Van den Wijngaard A.J. Pentenga M. Janssen D.B. Eur. J. Biochem. 1991; 202: 1217-1222Crossref PubMed Scopus (71) Google Scholar, 29Visser H. J Bont A.M.D. Verdoes J.C. Appl. Environ. Microbiol. 1999; 65: 5459-5463Crossref PubMed Google Scholar, 30Visser H. Vreugdenhil S. J Bont A.M.D. Verdoes J.C. Appl. Microbiol. Biotechnol. 2000; 53: 415-419Crossref PubMed Scopus (40) Google Scholar, 31Misawa E. Chion C.K.C.K. Archer I.V. Woodland M.P. Zhou N.Y. Carter S.F. Widdowson D.A. Leak D.J. Eur. J. Biochem. 1998; 253: 173-183Crossref PubMed Scopus (47) Google Scholar, 32Nakamura T. Nagasawa T. Yu F. Watanabe I. Yamada H. Appl. Environ. Microbiol. 1994; 60: 4630-4633Crossref PubMed Google Scholar), and various directed evolution and rational protein engineering techniques may be used to alter enzymatic activity (33Brannigan J.A. Wilkinson A.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 964-970Crossref PubMed Scopus (114) Google Scholar, 34Stemmer W.P.C. Nature. 1994; 370: 389-391Crossref PubMed Scopus (1639) Google Scholar). Hence, it was investigated here whether an epoxide hydrolase could be tuned to accept chlorinated epoxyethanes as a substrate. The EH from Agrobacterium radiobacter AD1 (EchA, Gen-Bank™ accession number Y12804) (35Rink R. Fennema M. Smids M. Dehmel U. Janssen D.B. J. Biol. Chem. 1997; 272: 14650-14657Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) was chosen for protein engineering because its physiological substrate, epichlorohydrin (2-chloropropylene oxide), resembles chlorinated epoxyethanes. EchA (294 amino acids) contains a core domain with typical α/β hydrolase fold topology formed by an eight-stranded β-sheet sandwiched by α-helices and an α-helical cap domain protruding from the core domain (36Nardini M. Ridder I.S. Rozeboom H.J. Kalk K.H. Rink R. Janssen D.B. Dijkstra B.W. J. Biol. Chem. 1999; 274: 14579-14586Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The catalytic triad residues Asp-107, Asp-246, and His-275 are located in a hydrophobic internal cavity between the two domains (36Nardini M. Ridder I.S. Rozeboom H.J. Kalk K.H. Rink R. Janssen D.B. Dijkstra B.W. J. Biol. Chem. 1999; 274: 14579-14586Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The catalytic reaction follows a two-step mechanism involving an alkyl-enzyme ester intermediate, which is further hydrolyzed via the attack of a water molecule (36Nardini M. Ridder I.S. Rozeboom H.J. Kalk K.H. Rink R. Janssen D.B. Dijkstra B.W. J. Biol. Chem. 1999; 274: 14579-14586Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 37Nardini M. Rink R. Janssen D.B. Dijkstra B.W. J. Mol. Catal. B Enzym. 2001; 11: 1035-1042Crossref Scopus (23) Google Scholar). We reasoned that the substrate range of the enzyme may be tailored to accept a chlorinated epoxyethane based on the three-dimensional structure of EchA (PDB accession code 1EHY) (36Nardini M. Ridder I.S. Rozeboom H.J. Kalk K.H. Rink R. Janssen D.B. Dijkstra B.W. J. Biol. Chem. 1999; 274: 14579-14586Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), an understanding of the molecular level properties of this enzyme (36Nardini M. Ridder I.S. Rozeboom H.J. Kalk K.H. Rink R. Janssen D.B. Dijkstra B.W. J. Biol. Chem. 1999; 274: 14579-14586Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), and its relatedness to similar EH enzymes (2Zou J. Hallberg B.M. Bergfors T. Oesch F. Arand M. Mowbray S.L. Jones T.A. Structure. 2000; 8: 111-122Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 38Argiriadi M.A. Morisseau C. Hammock B.D. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10637-10642Crossref PubMed Scopus (208) Google Scholar) and haloalkane dehalogenase (DhlA) (39Franken S.M. Rozeboom H.J. Kalk K.H. Dijkstra B.W. EMBO J. 1991; 10: 1297-1302Crossref PubMed Scopus (182) Google Scholar, 40Verschueren K.H.G. Seljee F. Rozeboom H.J. Kalk K.H. Dijkstra B.W. Nature. 1993; 363: 693-698Crossref PubMed Scopus (422) Google Scholar). Saturation mutagenesis was used rather than site-directed mutagenesis to introduce all possible mutations at one site to explore a larger fraction of the protein sequence space (41Sakamoto T. Joern J.M. Arisawa A. Arnold F.H. Appl. Environ. Microbiol. 2001; 67: 3882-3887Crossref PubMed Scopus (67) Google Scholar). This is the first report of targeted mutagenesis of epoxide hydrolases at these positions, of an epoxide hydrolase with activity toward chlorinated epoxyethanes, and of enhancement of cis-DCE degradation by combining an evolved monooxygenase and evolved epoxide hydrolase to detoxify the reactive intermediates. Chemicals, Organisms, and Growth Conditions—All materials were of highest purity available and purchased from Fisher Scientific Company (Pittsburgh, PA) except for epichlorohydrin (Acros Organics, Morris Plains, NJ), betaine (Sigma), and cis-DCE (TCI America, Inc., Portland, OR). E. coli TG1 (42.Gibson, T. J. (1984) Studies on the Epstein-Barr Virus Genome. Ph.D. thesis, Cambridge University, Cambridge, UKGoogle Scholar) was used for cloning and gene expression. Recombinant strains were routinely grown at 37 °C in Luria-Bertani (LB) broth (43Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) supplemented with kanamycin (Kan, 100 μg/ml) and chloramphenicol (Cam, 50 μg/ml) to maintain plasmids unless otherwise stated. All whole-cell experiments used LB + Kan + Cam cultures inoculated from single, fresh colonies; exponential phase cells were harvested at an optical density at 600 nm (A) of ∼1.5. Isopropyl β-d-thiogalactopyranoside (IPTG, 0.5 mm) was used to induce TOM-Green that was under control of the tac-lacUV5 tandem promoter in plasmid pMMB206 (44Morales V.M. Backman A. Bagdasarian M. Gene. 1991; 97: 39-47Crossref PubMed Scopus (427) Google Scholar) and also to induce EchA under control of the lac promoter in pBS(Kan) (26Canada K.A. Iwashita S. Shim H. Wood T.K. J. Bacteriol. 2002; 184: 344-349Crossref PubMed Scopus (154) Google Scholar); IPTG was added at an A of 0.2–0.3 for 2 h. The exponentially grown cells were washed three times with one volume of Tris-HNO3 buffer (50 mm, pH 7.0) to remove interfering byproducts and trace chloride (26Canada K.A. Iwashita S. Shim H. Wood T.K. J. Bacteriol. 2002; 184: 344-349Crossref PubMed Scopus (154) Google Scholar). Protein Analysis and Molecular Techniques—Total cellular protein for the exponentially growing culture was determined with the Total Protein kit (Sigma), and expression of recombinant proteins was analyzed with standard Laemmli discontinuous sodium dodecyl sulfate-12% polyacrylamide gels (SDS-PAGE) (43Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Plasmid DNA was isolated using a Midi or Mini kit (Qiagen, Inc.), and polymerase chain reaction (PCR) products were purified with a Wizard® PCR Preps DNA purification system (Promega Corp., Madison, WI). DNA fragments were isolated from agarose gels using a QIAquick gel extraction kit (Qiagen, Inc.). E. coli was transformed using electroporation with a Gene Pulser/Pulse Controller (Bio-Rad) at 15 kV/cm, 25 μF, and 200 Ω. PCR Amplification and Plasmid Construction—To stably and constitutively express the EH from A. radiobacter AD1, the echA gene was amplified by PCR using plasmid pEH20 (35Rink R. Fennema M. Smids M. Dehmel U. Janssen D.B. J. Biol. Chem. 1997; 272: 14650-14657Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) as the template with the forward primer 5′-ATAGCGGTACCACAACGGTTTCCCT-3′ and reverse primer 5′-ATTGCTGTCGACCAGTCATGCTAGCC-3′, where underlining indicates the KpnI and SalI restriction enzyme sites, respectively. The PCR amplification was performed with Pfu DNA polymerase (Stratagene) using a PCR program of 30 cycles of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 2 min, and a final extension of 72 °C for 10 min. The PCR fragment was double digested with KpnI and SalI and ligated into pBS(Kan) at the same restriction sites, yielding pBS(Kan)EH (Fig. 2). The six genes, tomA0-tomA5, of TOM-Green were obtained from plasmid pBS(Kan)TOM-Green (26Canada K.A. Iwashita S. Shim H. Wood T.K. J. Bacteriol. 2002; 184: 344-349Crossref PubMed Scopus (154) Google Scholar) after EcoRI and PvuI restriction digestion and purification from an agarose gel. The resulting 5345-bp fragment was ligated into pMMB206 after digestion with the same restriction enzymes, resulting in pMMB206-TOM-Green (Fig. 2). Saturation Mutagenesis of EchA—A gene library encoding all possible amino acids at positions Phe-108, Ile-111, Ile-219, and Cys-248 of EchA in pBS(Kan)EH was constructed by replacing the target codon with NNN via overlap extension PCR (41Sakamoto T. Joern J.M. Arisawa A. Arnold F.H. Appl. Environ. Microbiol. 2001; 67: 3882-3887Crossref PubMed Scopus (67) Google Scholar). Four pairs of degenerate primers, Phe-108 Front/Phe-108 Rear, Ile-111 Front/Ile-111 Rear, Ile-219 Front/Ile-219 Rear, and Cys-248 Front/Cys-248 Rear (Table I) were designed to randomize codons Phe-108, Ile-111, Ile-219, and Cys-248 in the nucleotide sequence, respectively. Two additional primers for cloning were EH Front and EH Rear (Table I), which were upstream and downstream of the natural KpnI and SacI restriction sites flanking the echA gene (Fig. 2). To minimize random point mutations, Pfu DNA polymerase was used in the PCR. Addition of betaine (1 m) in the PCR mixture was used to improve the amplification of DNA by reducing the formation of secondary structure in the GC-rich region when necessary (45Baskaran N. Kandpal R.P. Bhargava A.K. Glynn M.W. Bale A. Weissman S.M. Genome Res. 1996; 6: 633-638Crossref PubMed Scopus (142) Google Scholar). In the first round of saturation mutagenesis, pBS(Kan)EH was used as the template, and mutagenesis was performed individually at sites Phe-108, Ile-111, Ile-219, and Cys-248. In the second round of mutagenesis, pBS(Kan)EH C248I (containing amino acid substitution Cys-248I in EchA) was used as the template and sites Phe-108 and Ile-219 were randomized individually. In the third round, pBS(Kan)EH F108L/C248I (containing amino acid substitutions F108L and C248I in EchA) was used as the template and site Ile-219 was subjected to saturation mutagenesis. Two degenerate PCR fragments were produced for each site with 463 and 749 bp for site Phe-108, 457 and 754 bp for site Ile-111, 800 and 414 bp for site Ile-219, and 853 and 327 bp for site Cys-248. After purifying from agarose gels, the two fragments for each site were combined at a 1:1 ratio as templates to obtain the full-length PCR product with the EH Front and EH Rear primers. The resulting randomized PCR product (1167 bp) was cloned into pBS(Kan)EH after double digestion with KpnI and SacI, replacing the corresponding fragment in the original plasmid.Table IOligonucleotide primers used for saturation mutagenesis at positions Phe-108, Ile-111, Ile-219, and Cys-248 of EchAPrimerSequencesPhe-108 FrontAGGCGTACGTCGTTGGCCATGACNNNGCGGCCATCPhe-108 RearTTATGGAGGACGATGGCCGCGNNNTCATGGCCAACIle-111 FrontACGTCGTTGGCCATGACTTCGCGGCCNNNGTCCTCCIle-111 RearATGAATTTATGGAGGACNNNGGCCGCGAAGTCATGIle-219 FrontCTTCAACTACTATCGTGCCAACNNNAGGCCCGATGIle-219 RearACAGAGCGGCATCGGGCCTNNNGTTGGCACGATAGCys-248 FrontATATGGGGTTTGGGAGATACTNNNGTGCCCTATGCCys-248 RearTCAATGAGTGGAGCATAGGGCACNNNAGTATCTCCEH FrontaUtilizing the natural KpnI restriction site upstream of echA.AGCTATGACCATGATTACGCCAAGCEH RearbUtilizing the natural SacI restriction site downstream of echA.CGTTGTAAAACGACGGCCAGTGAa Utilizing the natural KpnI restriction site upstream of echA.b Utilizing the natural SacI restriction site downstream of echA. Open table in a new tab Screening for Enhanced cis-DCE Degradation—Evolved EchA activity toward cis-DCE epoxide was found indirectly by monitoring the concentration of chloride ion released from cis-DCE epoxide (generated by TOM-Green oxidation of cis-DCE) degradation by the evolved EchA (Fig. 1). TG1 cells harboring plasmids pMMB206-TOM-Green and pBS(Kan)EH variants were grown in 96-well plates, washed three times with Tris-HNO3 buffer (50 mm, pH 7.0), and contacted with shaking at 37 °C in an airtight chamber, 23 × 20 × 23 cm, with cis-DCE vapor (2 ml) for 18 h. The inorganic chloride ions generated from the mineralization of cis-DCE by whole cells were detected by adding 40 μl of 0.25 m Fe(NH4)(SO4)2 in 9 m HNO3 and 40 μl of saturated Hg(SCN)2 in 95% ethanol to the 200 μl of supernatant in each well of the 96-well plate and measured at 450 nm (26Canada K.A. Iwashita S. Shim H. Wood T.K. J. Bacteriol. 2002; 184: 344-349Crossref PubMed Scopus (154) Google Scholar). Extent and Kinetics of cis-DCE Mineralization—For determining the extent of mineralization of cis-DCE (as indicated by Cl– production) from the best colonies identified by the 96-well screening, the exponentially growing cells were washed three times with Tris-HNO3 buffer (50 mm, pH 7.0). Then the cell suspension (2.5 ml) was adjusted to an A of 3, sealed in 15-ml glass serum vials, and contacted with cis-DCE at an initial liquid concentration of 540 μm (based on a Henry's Law constant of 0.17 (46Dolfing J. van den Wijngaard A.J. Janssen D.B. Biodegradation. 1993; 4: 261-282Crossref PubMed Scopus (63) Google Scholar)). 2.5 μmol of cis-DCE was injected into the cells in 5 μl of dimethyl formamide (DMF) at 0.2 vol%. Isopropyl β-d-thiogalactopyranoside (0.5 mm) was added along with 5 mm sodium succinate (as a substrate to produce NADH). After 2 h of incubation at 37 °C and 250 rpm, the whole-cell reaction was quenched by heating the vials in boiling water for 90 s and centrifuging (16,000 × g, 4 min) to collect the supernatant. Chloride ion concentrations in 500 μl of supernatant were measured spectrophotometrically at 460 nm as indicated above. Cells contacted with the same amount of DMF were used as the negative control, and at least three independent experiments were analyzed. To determine the kinetics of cis-DCE mineralization, the A of the cell culture was 1.2, and the initial cis-DCE concentrations were 6.8 to 540 μm (using different stock solutions of 6.25, 25, 125, and 500 mm in DMF at 0.2–0.4 vol%). The supernatant chloride ion concentrations generated from mineralizing cis-DCE for each concentration were measured at 9 min for 6.8 and 13.5 μm, 15 min for 27 μm, 21 min for 54 μm, 38 min for 135 μm, 56 min for 270 μm, and 67 min for 540 μm. The contacting times were varied to detect significant Cl– while maintaining the mineralization rate in the linear range for each cis-DCE concentration. Parallel experiments determining the cis-DCE degradation rate were conducted using gas chromatography (GC) to monitor cis-DCE depletion as described previously (25Shim H. Wood T.K. Biotechnol. Bioeng. 2000; 70: 693-698Crossref PubMed Scopus (33) Google Scholar). Headspace samples from the same cell suspensions contacted with cis-DCE at various concentrations in the cis-DCE kinetics experiments were analyzed before the cells were quenched to determine Cl– production. Purification of EchA—Both wild-type EchA and variant F108L/I219L/C248I were expressed in TG1/pBS(Kan) for enzyme purification, and the method of Rink et al. (35Rink R. Fennema M. Smids M. Dehmel U. Janssen D.B. J. Biol. Chem. 1997; 272: 14650-14657Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) was adopted with modification. Exponentially growing precultures were diluted 1:100 in 3 liters of fresh Luria-Bertani broth containing 100 μg/ml Kan and incubated with shaking at 37 °C. Isopropyl β-d-thiogalactopyranoside (1 mm) was added when the cell A reached 0.3, and the culture was incubated at 25 °C overnight with aeration. Cells were harvested, washed by centrifugation at 10,000 × g, 4 °C for 10 min with TEMAG buffer (10 mm Tris-SO4, 1 mm EDTA, 1 mm β-mercaptoethanol, 0.02% sodium azide, and 10% glycerol, pH 7.5), and resuspended in 30 ml of the same buffer. Cells were disrupted by a French® pressure cell press (Spectronic Instruments, Rochester, NY) and centrifuged at 20,000 × g, 4 °C for 30 min. Anion exchange was performed by applying 30 ml of the supernatant to 30 ml of DEAE-Sepharose (Sigma) (47Spelberg J.H.L. Rink R. Kellogg R.M. Janssen D.B. Tetrahedron Asymmetry. 1998; 9: 459-466Crossref Scopus (90) Google Scholar), and proteins were eluted with a continuous gradient ammonium sulfate in TEMAG (0–1 m). Fractions with the highest enzymatic activity were pooled and dialyzed against TEMAG buffer overnight at 4 °C and were further purified via size exclusion chromatography by adding 1.5 ml to 80 ml of Sephacryl S-100 HR (Sigma). Fractions with purified EchA (with the highest EH activity and the highest purity as visualized on SDS-PAGE) were pooled and dialyzed against TEMAG buffer overnight. The final product was stored at –20 °C with glycerol (10% v/v) for future use; variant F108L/I219L/C248I and wild-type enzyme were purified from 10% to 80 and 90%, respectively. Activity of column fractions was determined using a polypropylene 96-well plate format with styrene oxide (5 mm) as the substrate rather than the reported epichlorohydrin (35Rink R. Fennema M. Smids M. Dehmel U. Janssen D.B. J. Biol. Chem. 1997; 272: 14650-14657Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Column fractions (10 μl) were added to 136 μl of TE buffer and incubated with 5 mm styrene oxide at 37 °C for 15 min, followed by the sequential addition of 100 mm 4-nitrobenzylpyridine (75 μl) and triethylamine (75 μl). The chromogenic reaction of styrene oxide with 4-nitrobenzylpyridine was measured at 620 nm with a Multiskan reader (Fisher Scientific), and disappearance of the blue color indicated the disappearance of styrene oxide. EH Assays—As a preliminary assay, whole cells of TG1/pBS(Kan)EH (grown in Luria-Bertani broth + 100 μg/ml Kan) were tested for EchA activity in E. coli using a chromogenic reaction of the epoxide epichlorohydrin with 4-nitrobenzylpyridin (35Rink R. Fennema M. Smids M. Dehmel U. Janssen D.B. J. Biol. Chem. 1997; 272: 14650-14657Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The assay was performed in 1.5-ml microcentrifuge tubes with 100 μl of exponentially grown cells contacted with 10 mm epichlorohydrin in 400 μl of TE buffer (50 mm Tris-SO4, 1 mm EDT

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