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

Abstract

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.

Fig. 1.

Three POA binding sites found in the active tunnel of the BmEH–POA complex. (A) POA molecules in zones 1 (yellow), 2 (green), and 3 (cyan). (B–D) The POA binding pattern in zones 1 (B), 2 (C), and 3 (D). The residues of BmEH interacting directly with POA are shown as ball-and-stick models.

Fig. 2.

Mass-spectrometric analysis of the covalent intermediates in the BmEH-catalyzed NGE hydrolysis reaction. (A) Relative activities of mutant M145F and T241R/L168E compared with the wild-type BmEH. (B–D) Comparison of the molecular mass changes of the H267F (B), T241R/L168E/H267F (C), and M145F/H267F (D) BmEH variants before (black) and after (red) reacting with NGE. The theoretical molecular mass increase of the enzymes is 200 Da. Mutations at residue Met-145 severely blocked the formation of the enzymatic covalent intermediates.

Fig. 3.

The enzyme–substrate intermediate formation curve of H267F (A), F128A/H267F (B), and M145A/H267F (C). ●, Concentration of substrate (NGE); ■, concentration of product (NPD); ▲, concentration of intermediate. Values were calculated by deduction of the remaining NGE and NPD concentrations from the inputs (25 μM). Reaction conditions were as follows: Each variant at a concentration of 25 μM was mixed with 25 μM (R)-NGE in potassium phosphate buffer (100 mM, pH 7.0) containing 10% DMSO at 30 °C. Samples were withdrawn at different intervals, mixed with methanol for termination, and analyzed by RP-HPLC (C18 column). The concentration of NGE and NPD was quantified by the area of corresponding peaks on HPLC.

Fig. 4.

The M145A and F128A mutations made zone 2 expand and attach to zone 3. (A−C) Surface presentation of zones 2 and 3 in wild-type BmEH (cyan), M145A (yellow), and F128A (gray). (D) Snapshots of MD simulation on the BmEH_F128A–(R)-NPD complex at 0 ns (green), 42 ns (blue), and 80 ns (yellow). Significant conformation changes took place in residues Trp-98, Leu-132, and Phe-209 around zones 2 and 3.

Fig. 5.

A proposed model for bulky (R)-diol production by the M145A and F128A BmEH variants. The M145A or F128A mutation expanded the predicted product release site of BmEH, which would facilitate the hydrolysis of bulky NGE. Tyrosines (Tyr-144 and -203) and aspartic acid (Asp-97) were served as Lewis acid and Lewis base, respectively.