Structure-Guided Engineering of d-Carbamoylase Reveals a Key Loop at Substrate Entrance Tunnel

A d-carbamoylase NiHyuC from Nitratireductor indicus was identified with high catalytic activity toward N-carbamoyl-d-tryptophan (3a). To further enhance its efficiency, both random mutagenesis and structure-guided evolution were performed. Variant M4 (D187N/A200N/S207A/R211G) showed a 43-fold increase in catalytic efficiency (kcat/Km = 1135.0 min–1 mM–1) and a 21-fold reduction in Km value (0.4 mM) compared with WT. Crystal structures of beneficial variants were resolved to clarify the evolutionary changes underlying improvements to catalytic efficiency. Structure alignment with WT indicated that loop 200–207 may play an important role in modulating access to the substrate entrance tunnel. Furthermore, MD simulations of WT–3a and M4–3a interactions illustrated that M4–3a has a better angle for nucleophilic attack and more readily enters a prereaction state. Additional hydrogen bonds and hydrophobic interactions were observed in prereaction states of M4–3a compared with that of WT–3a, consistent with its decreased Km value. In a hydantoinase process, the complete conversion of 160 mM l-indolylmethylhydantoin was achieved by M4 in a 0.5 L reaction, with d-tryptophan yield of 99.3% and productivity of 64.9 g L–1 d–1. This study reveals a key loop at the substrate tunnel of d-carbamoylase and provides an effective strategy for engineering d-carbamoylase and other carbon–nitrogen hydrolase family enzymes.