Remarkable improvement in the regiocomplementarity of a Glycine max epoxide hydrolase by reshaping its substrate-binding pocket for the enantioconvergent preparation of (R)-hexane-1,2-diol

E. coli/gmeh3, an E. coli transformant expressing GmEH3, had the best regiocomplementarity (alpha(S) = 72.4% and beta(R) = 97.6%) for (S)-and (R)-1,2-epoxyhexanes among five tested aliphatic chain rac-1,2-epoxides (1a-5a). To prepare (R)-hexane-1,2-diol (1b) with high ee(p) via enantioconvergent hydrolysis of rac-1a, the regioselectivity coefficient alpha(S) of GmEH3 for (S)-1a was improved by reshaping its substrate-binding pocket (SBP). Based on the semi-rational design, Trp(102), Ile(105), Ile(178), Pro(187) and Leu(189) lining GmEH3's SBP were identified, each of which was substituted with four residues, respectively. From 17 transformants harboring single-site variants of gmeh3, E. coli/gmeh3(W102L), /gmeh3(W102I), /gmeh3(W102V) and /gmeh3(P187F) were selected, catalyzing the conversion of rac-1a into (R)-1b with obviously enhanced ee(p) values of 59.3-78.3%. Then, three transformants containing double-site variants were constructed by combinatorial mutagenesis of gmeh3(P187F) separately with gmeh3(W102L), gmeh3(W102I) and gmeh3(W102V). Among the three transformants, E. coli/gmeh3(W102V/P187F) displayed the largest alpha(S) of 89.7% with beta(R) of 96.2%. The enantioconvergent hydrolysis of 500 mM rac-1a was conducted using 200 mg/mL wet cells of E. coli/gmeh3(W102V/P187F) at 25 degrees C for 12 h, affording (R) -1b with 83.1% ee(p) and 91.5% yield. The molecular docking simulation analysis demonstrated that GmEH3(W102V/P187F) more regiopreferentially attacks C-alpha in the oxirane ring of (S)-1a than GmEH3.

Automated summary

In this study, regioselectivity of GmEH3 for (S)-1a was improved by reshaping its substrate-binding pocket (SBP), leading to the development of E. coli transformants capable of efficiently synthesizing (R)-1b. Mutagenesis was used to enhance the enzyme's selectivity towards the (R)-1b product, laying the foundation for the synthesis of (R)-hexane-1,2-diol with high ee values.