Manipulating the regioselectivity of aSolanum lycopersicumepoxide hydrolase for the enantioconvergent synthesis of enantiopure alkane- and alkene-1,2-diols

摘要

The hydrolysis of epoxides by epoxide hydrolases (EHs) is a sustainable approach to synthesize chiral vicinal diols. However, few EHs hitherto reported can catalyse the enantioconvergent synthesis of enantiopure alkane- and alkene-1,2-diols, due to the strict desire for regiocomplementarity. To actualize the enantioconvergent hydrolysis ofrac-1,2-epoxyoctane (1a), the regioselectivity (alpha(S), 55.3%) ofSlEH1 for (S)-1awas manipulatedviasite-directed mutagenesis. Ten specific residues lining the substrate-binding pocket ofSlEH1 were identified based on the computer-aided design, each of which was replaced by seven residues, respectively. Among 66 single-site mutants,SlEH1(W106L),SlEH1(W106T),SlEH1(F109I),SlEH1(M180V),SlEH1(F189I)andSlEH1(F189L)were selected, by whichrac-1awas hydrolysed into (R)-octane-1,2-diol (1b) with an ee(p)range of 62.1-82.4%. After combinatorial mutagenesis and screening, one double-site mutant,SlEH1(W106T/F189L), was obtained having the highest alpha(S)of 96.7%. The gram-scale enantioconvergent hydrolysis of 400 mM (51.3 g L-1)rac-1awas carried out using 200 mg mL(-1)wet cells ofE. coli/sleh1(W106T/F189L)at 20 degrees C for 24 h, producing (R)-1bwith 94.7% ee(p)and 95.6% yield. The substrate spectrum assay ofSlEH1(W106T/F189L)towards 20 mMrac-1a-6awas conducted, producing (R)-1b-6bwith 81.1-97.4% ee(p)values. Furthermore, the molecular dynamics simulation analysis indicated that the regiopreference ofSlEH1(W106T/F189L)attacking on the C(alpha)of (S)-1awas greater than that ofSlEH1, which was consistent with their alpha(S)values measured experimentally. This work engineered a superior double-site mutant,SlEH1(W106T/F189L), for the enantioconvergent synthesis of a variety of chiral alkane- and alkene-1,2-diols, especially (R)-1band6b, with high ee(p)values.