Title: Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling van Loo B, Spelberg JH, Kingma J, Sonke T, Wubbolts MG, Janssen DB Ref: Chemical Biology, 11:981, 2004 : PubMed
The enantioselectivity of epoxide hydrolase from Agrobacterium radiobacter (EchA) was improved using error-prone PCR and DNA shuffling. An agar plate assay was used to screen the mutant libraries for activity. Screening for improved enantioselectivity was subsequently done by spectrophotometric progress curve analysis of the conversion of para-nitrophenyl glycidyl ether (pNPGE). Kinetic resolutions showed that eight mutants were obtained with up to 13-fold improved enantioselectivity toward pNPGE and at least three other epoxides. The large enhancements in enantioselectivity toward epichlorohydrin and 1,2-epoxyhexane indicated that pNPGE acts as an epoxyalkane mimic. Active site mutations were found in all shuffled mutants, which can be explained by an interaction of the affected amino acid with the epoxide oxygen or the hydrophobic moiety of the substrate. Several mutations in the shuffled mutants had additive effects.
The detoxication of the enantiomers of glycidyl 4-nitrophenyl ether (GNPE), (-)-(R)- and (+)-(S)-GNPE, and glycidyl 1-naphthyl ether (GNE), (-)-(R)- and (+)-(S)-GNE, by rat liver glutathione transferase and epoxide hydrolase was studied. Enantioselectivity was observed with both enzymes favoring the (R)-isomers as determined by the formation of conjugate, diol, and remaining substrate measured by HPLC. Enantiomers of GNE were detoxified by cytosolic epoxide hydrolase but those of GNPE were not. Substantial nonenzymatically formed conjugates of enantiomers of GNPE were detected showing (S)-GNPE the more reactive of the pair.
Two convenient and sensitive continuous spectrophotometric assays for cytosolic epoxide hydrolase are described. The assays are based on the differences in the ultraviolet spectra of the epoxide substrates and their diol products. The hydrolysis of 1,2-epoxy-1-(p-nitrophenyl)pentane (ENP5) is accompanied by a decrease in absorbance at 302 nm, while the hydration of 1,2-epoxy-1-(2-quinolyl)pentane (EQU5) produces an increase in absorbance at 315.5 nm. The Km, Vmax values for ENP5 and EQU5 with purified mouse liver cytosolic epoxide hydrolase were 1.7 microM, 11,700 nmol/min/mg and 25 microM, 8300 nmol/min/mg, respectively. Both substrates are hydrolyzed significantly faster than trans-stilbene oxide, which is currently the most commonly used substrate for measuring cytosolic epoxide hydrolase activity. No spontaneous hydrolysis of the substrates is detectable under normal assay conditions. The assays are applicable to whole tissue homogenates as well as purified enzyme preparations. p-Nitrostyrene oxide and p-nitrophenyl glycidyl ether were also examined and found to be very poor substrates for cytosolic epoxide hydrolase from mouse liver.
