Two alternative modes for optimizing nylon‐6 byproduct hydrolytic activity from a carboxylesterase with a β‐lactamase fold: X‐ray crystallographic analysis of directly evolved 6‐aminohexanoate‐dimer hydrolase

Promiscuous 6‐aminohexanoate‐linear dimer (Ald)‐hydrolytic activity originally obtained in a carboxylesterase with a β‐lactamase fold was enhanced about 80‐fold by directed evolution using error‐prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb‐S4M94) demonstrated that the enzyme had acquired an increased affinity (K_m = 15 mM) and turnover (k_cat = 3.1 s⁻¹) for Ald, and that a catalytic center suitable for nylon‐6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb‐S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen‐bonding/hydrophobic interactions between the AldCOOH and Tyr370, a hydrogen‐bonding network from Ser187 to AldNH, and interaction between AldNH and Gln27‐O_ε derived from another subunit in the homo‐dimeric structure. In wild‐type Ald‐hydrolase (NylB), Ald‐hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with AldNH (Kawashima et al., FEBS J 2009; 276:2547–2556). We propose here that there exist at least two alternative modes for optimizing the Ald‐hydrolytic activity of a carboxylesterase with a β‐lactamase fold.