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 groundwater 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 wildtype EchA at low concentrations (6.8 M) and up to 10-fold at high concentrations (540 M). EchA variants with single mutations (F108L, I219F, or C248I) enhanced cis-DCE mineralization 2.5-fold (540 M), and EchA variants with double mutations, I219L/C248I and F108L/C248I, increased cis-DCE mineralization 4-and 7-fold, respectively (540 M). For complete degradation of cis-DCE to chloride ions, the apparent V max /K m for the recombinant Escherichia coli strain expressing 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).

Living biofouling-resistant membranes as a model for the beneficial use of engineered biofilms

“…A chromogenic reaction of epoxide epichlorohydrin with 4-nitrobenzylpyridine was used to measure the activity of EH (67) using planktonic cells. The assay was performed in 1.5-mL microcentrifuge tubes as described previously (44). Diluted overnight cultures at an initial turbidity at 600 nm of 0.05 were inoculated in LB with 1% arabinose at 37°C.…”

Protein Engineering of Epoxide Hydrolase from Agrobacterium radiobacter AD1 for Enhanced Activity and Enantioselective Production of ( R )-1-Phenylethane-1,2-Diol

“…Expression of recombinant proteins was analyzed by standard Laemmli discontinuous SDS-PAGE (12%) (28). Purification of recombinant proteins was conducted using the method of Rink et al (21) with the modifications described previously (25).…”

“…Escherichia coli strain TG1 (8) was utilized as the host for cloning and functional expression of genes. Recombinant strains were routinely cultivated at 37°C in Luria-Bertani (LB) broth (28) supplemented with kanamycin (Kan; 100 g/ml) to maintain plasmid pBS(Kan)EH (25), which constitutively expresses EchA from a lac promoter. All experiments (unless otherwise stated) were conducted using exponential-phase cultures in LB-plus-Kan medium which were started from fresh colonies, induced fully with 1.0 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) at an optical density at 600 nm (OD 600 ) of 0.2 to 0.3, and harvested after 2 h of IPTG induction with an OD 600 of 1.5 to 2.0 (IPTG induction was used to ensure equal, high expression of the EchA isoforms).…”

“…Previously, the EchA active-site residues F108, I219, and C248 were altered to enhance the degradation of cis-1,2-dichloroethylene epoxide (cis-DCE epoxide) (25). The variants created in that study (25) were evaluated here for altered enantioselectivity toward racemic styrene oxide, which has been used as a model substrate for studying the enantioselectivity of EchA (22,31).…”

“…The variants created in that study (25) were evaluated here for altered enantioselectivity toward racemic styrene oxide, which has been used as a model substrate for studying the enantioselectivity of EchA (22,31). Further, DNA shuffling was used to isolate EchA variants with altered enzymatic activity and enantioselectivity toward racemic styrene oxide, so that residues, especially those having a far-reaching effect on substrate binding that are not reliably predicted by rational design, may be identified; and saturation mutagenesis was subsequently used to evaluate all amino acids at the important sites identified from random mutagenesis in this study.…”