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

Proc Natl Acad Sci U S A. 2014 Nov 4;111(44):15717-22. doi: 10.1073/pnas.1404915111. Epub 2014 Oct 20.


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 BmEH, 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 BmEH 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.

Keywords: X-ray crystallography; bulky substrate; epoxide hydrolase; product release; protein engineering.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adrenergic beta-Antagonists / chemical synthesis
  • Adrenergic beta-Antagonists / chemistry*
  • Alprenolol / chemical synthesis
  • Alprenolol / chemistry*
  • Amino Acid Substitution
  • Bacillus megaterium / enzymology*
  • Bacillus megaterium / genetics
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Crystallography, X-Ray
  • Epoxide Hydrolases / chemistry*
  • Epoxide Hydrolases / genetics
  • Epoxy Compounds / chemistry
  • Mutation, Missense
  • Naphthols / chemistry
  • Propranolol / chemical synthesis*
  • Propranolol / chemistry


  • Adrenergic beta-Antagonists
  • Bacterial Proteins
  • Epoxy Compounds
  • Naphthols
  • glycidyl 1-naphthyl ether
  • Alprenolol
  • Propranolol
  • Epoxide Hydrolases

Associated data

  • PDB/4G00
  • PDB/4G02
  • PDB/4INZ
  • PDB/4IO0