Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

Optically pure epoxides are essential chiral precursors for the production of ( S )-propranolol, ( S )-alprenolol, and other β-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of Bm EH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of ( S )-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in Bm EH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward α-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates. Significance Application of epoxide hydrolases in synthesizing chiral drug compounds has been hindered by their limited substrate range. The enzymatic production of bulky epoxides has proven remarkably challenging. In this work, we identified an active tunnel for substrate access and product release of an epoxide hydrolase with unusual ( R )-enantioselectivity. Mutagenesis targeted to unblock the steric hindrance in the active pocket or the potential product release site resulted in variants with much higher activity toward α-naphthyl glycidyl ether, the precursor of β-adrenergic receptor blocking drug ( S )-propranolol. The strategy presented here may be a useful alternative choice for rational design of enzymes toward bulky substrates.