Rational design of d-hydantoinase from Pseudomonas aeruginosa for desymmetric synthesis of chiral intermediate for (S)-pregabalin

( S )-Pregabalin ( S -PGB) is extensively employed in analgesic therapy. One of the current chemical processes used to produce S -PGB involves the Hofmann rearrangement of ( R )-3-isobutyl glutaric acid monoamide ( R -IBM). However, the chemical resolution of R -IBM is inefficient and environmentally unsustainable. In this study, we established an enzyme-catalyzed route to R -IBM that proceeds with exceptional optical purity and yield. We successfully resculpted the substrate-binding pocket of d- hydantoinase by attenuating the steric hindrance and enhancing hydrophilicity, resulting in accelerated substrate transport and restricted side-chain rotation during the asymmetric hydrolysis process. Notably, both optical purity and yield for the generation of R -IBM from the prochiral substrate 3-isobutyl glutarimide (IBI) were enhanced for the M3 (L64A/F66H/C318T) mutant; the enantiomeric excess increased from 65.51 % ± 5.85 % to 98.62 %± 0.28 % (ee p ) and k cat / K m increased from 14.92 to 207.06 s −1 ∙M −1 . The ancestral sequence reconstruction (ASR) and consensus analyses guided further engineering of the flexible C-terminal region of d- hydantoinase to enhance the thermostability of the enzyme. The final mutant, M6 (L64A/F66H/C318T/R99G/A265P/G466N/E470A), demonstrated a remarkable improvement in R -IBM production efficiency; thus, 500 mM IBI was completely transformed into R -IBM in 24 h. This study therefore showcases a new enzyme that offers a green, enzymatic route for R -IBM production.