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

Abstract

• A superior double-site mutant of Gm EH3, Gm EH3 W102V/P187F , was obtained by semi-rational design. • The α S of Gm EH3 W102V/P187F for ( S )- 1a was obviously increased to 89.7% from 72.4% of Gm EH3. • Enantioconvergent hydrolysis of rac - 1a by E. coli / gmeh3 W102V/P187F gave ( R )- 1b with 83.1% ee p . E. coli / gmeh3 , an E. coli transformant expressing Gm EH3, had the best regiocomplementarity (α S = 72.4% and β 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 α S of Gm EH3 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 Gm EH3’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 α S of 89.7% with β 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°C for 12 h, affording ( R )- 1b with 83.1% ee p and 91.5% yield. The molecular docking simulation analysis demonstrated that Gm EH3 W102V/P187F more regiopreferentially attacks C α in the oxirane ring of ( S )- 1a than Gm EH3.