title: 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.
abstract: 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 microm) and up to 10-fold at high concentrations (540 microm). EchA variants with single mutations (F108L, I219F, or C248I) enhanced cis-DCE mineralization 2.5-fold (540 microm), and EchA variants with double mutations, I219L/C248I and F108L/C248I, increased cis-DCE mineralization 4- and 7-fold, respectively (540 microm). 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).
authors.affiliation: Department of Chemical Engineering, University of Connecticut, Storrs, Connecticut 06269-3222, USA.
authors.forename: Lingyun
authors.initials: L
authors.lastname: Rui
authors.forename: Li
authors.initials: L
authors.lastname: Cao
authors.forename: Wilfred
authors.initials: W
authors.lastname: Chen
authors.forename: Kenneth F
authors.initials: KF
authors.lastname: Reardon
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Wood
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D001665:Binding Sites; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D004000:Dichloroethylenes / Q000737:chemistry / Q000494:pharmacology*; D004811:Epichlorohydrin / Q000737:chemistry; D004851:Epoxide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D010101:Oxygen Consumption; D010649:Phenylalanine / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014965:X-Rays
chemical_list: D004000:Dichloroethylenes; D007532:Isoleucine; D004811:Epichlorohydrin; D010649:Phenylalanine; D006899:Mixed Function Oxygenases; C093480:toluene ortho-monooxygenase; D004851:Epoxide Hydrolases; D003545:Cysteine; D010100:Oxygen; C035384:1,2-dichloroethylene