Thermotolerant D-Hydantoinases for Efficient and Stereoselective Intermediate Synthesis of (R)-3-Isobutylglutaric...

Pregabalin is a widely used clinical pharmaceutical agent for treatment of neuropathic pain and as an adjunctive therapy for epilepsy. The efficient synthesis of (R)-3-isobutylglutaric acid monoamide (R-IBM), a critical intermediate, is crucial for pregabalin manufacturing. This present study focused on identifying R-selective and robust D-hydantoinases with R-selectivity and robustness for the stereoselective synthesis of R-IBM, a key intermediate of pregabalin, from 3-isobutylglutarimide (IBI). Three D-hydantoinases, namely GsDHTase (GenBank No. PX317655), AcDHTase (GenBank No. PX317656) and BhDHTase (GenBank No. PX317657) with unique catalytic advantages were successfully isolated from a marine gene resource library. These enzymes exhibited exceptional robustness under extreme conditions (pH 10, 70°C or 80°C) and inherent high stereoselectivity for R-IBM. Wild-type and mutant forms of the three D-hydantoinases were heterologously expressed in E. coli BL21(DE3) using the pET-28a vector, followed by whole-cell catalysis experiments. The optimal IPTG induction temperature was determined to be 28°C. Maximum catalytic activity, corresponding to the highest conversion rate of IBI to R-IBM, was achieved at 50°C and pH 10.0. Notably, triple amino acid mutations (M63A, F65H and C317T) significantly enhanced stereoselectivity with only a slight reduction in enzymatic activity. The enantiomeric excess (ee%) of R-IBM improved from 73.74% to 97.55% for GsDHTase, 56.08% to 97.59% for AcDHTase and 78.32% to 96.85% for BhDHTase after mutation. Owing to their inherent robustness, these enzymes hold great potential for developing a theoretical 100% atom-economical route for the eco-friendly and cost-effective industrial production of pregabalin.