| Epoxide hydrolases (EHs) stereroselectively catalyze the hydration of epoxides to their corresponding vicinal diols. A great number of EHs have been discovered with good regeoselectivity and enantioselectivity, which makes them ideal biocatalysts for the synthesis of enantiopure epoxides and diols. Up to now, hundreds of EHs have been reported, but their application in synthesis of various epoxides is limited by the high substrate specificity and low catalytic efficiency towards unnatural epoxides like the synthetic precursors of a group of cardiovascular β-blocker drugs. We have previously cloned a novel EH (BmEH) from Bacillus megaterium ECU1001with unusual (R)-enantioselectivity (E=58) and high activity of83U mg-1protein towards phenyl glycidyl ether, however, its activities toward other pharmaceutically relevant epoxides are much lower (i.e. decreased by two magnitudes for bulky a-naphthyl glycidyl ether, NGE), which greatly limits the application of EHs for synthesis of chiral drugs. The aim of this research is to reveal the structure-function relationship of BmEH and to redesign the active site of enzyme to expand the substrate profile of BmEH and to promote their application in pharmaceutical synthesis.We first solved the crystal strucutres of native BmEH at1.75A resolution and its complex structure with a substrate analogue at1.95A resolution. Using X-ray crystallography, mass spectrum and molecular dynamics calculation, we have identified an active tunnel of this enzyme for substrate access and product release. The steric hindrance near the potential product-release site was predicted as the main obstacle for BmEH to catalyze the hydrolysis of bulky epoxides. By alanine scanning of residues around the active site, two mutants F128A and M145A were targeted to expand the product-release site, which displayed42and26times higher activities towards a-naphthyl glycidyl ether, respectively, than the wild-type enzyme. By determining the crystal structure of BmEH variants and the efficiency of intermediate formation of BmEHwiid-type and variants, we proved that both the rates of intermediate formation and product release were improved by the active site mutations.Subsequently, starting from BmEH, we constructed a small but smart library of EH variants by redesigning the active site with varied steric hindrance and hydrophobicity at the two hot spots predicted, namely Met145and Phe128. The activity and enantioselectivity of BmEH variants towards PGE and9typical β-blocker precursors were determined, to profile their substrate spectra. Surprisingly, BmEH variants exhibited6-430folds of activity enhancement for β-blocker precursors as compared with BmEH wild-type, with no significant decrease or even some improvement in enantioselectivity. Kinetic results showed that the highest improvement of BmEH catalytic efficiency (kcat/KM) achieved by mutation was896-fold, which was mainly derived from the dramatically enhanced kcat of variants. Further synthetic application of BmEH variants in gram-scale (1-10g) bioresolution of6(β-blocker precursors were performed, affording their (S)-enantiomers with96.6-99.5%ee and37.3-44.6%yields.To verify the application potential of BmEH variants in drug synthesis, the (R)-and (S)-enantiomers of biologically active propranolol were prepared in high optical purity via enzymatic kinetic resolution of racemic NGE. A preparative resolution of100g L-1rac-NGE (200g L-1in organic phase) was accomplished with a high enantioselectivity (E=92) using whole cell of BmEHF128T as biocatalyst in an optimized two-phase system of water/isopropyl ether/isooctane (50/35/15, by volume). The space-time yield of the enantiopure (S)-NGE (>99%ee) and (R)-NPD [(R)-3-(1’-naphthyloxy)propane-1,2-diol,>99.5%ee] were136g L-d-1and139g L-1d-1, respectively.(R)-NPD and (S)-NGE were chemically converted to (R)-and (S)-propranolol (each>99%ee) in overall yields of31.4%and44.8%, respectively. |
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