title: Two alternative modes for optimizing nylon-6 byproduct hydrolytic activity from a carboxylesterase with a beta-lactamase fold: X-ray crystallographic analysis of directly evolved 6-aminohexanoate-dimer hydrolase.
abstract: Promiscuous 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a beta-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(3) (+), and interaction between Ald--NH(3) (+) and Gln27-O(epsilon) 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(3) (+) (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 beta-lactamase fold.
authors.affiliation: Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, Hyogo 671-2280, Japan.
authors.forename: Taku
authors.initials: T
authors.lastname: Ohki
authors.forename: Naoki
authors.initials: N
authors.lastname: Shibata
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Higuchi
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Kawashima
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takeo
authors.forename: Dai-Ichiro
authors.initials: D
authors.lastname: Kato
authors.forename: Seiji
authors.initials: S
authors.lastname: Negoro
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000502:physiology; D001173:Arthrobacter / Q000201:enzymology; D002209:Caprolactam / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D043182:Carboxylesterase / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006868:Hydrolysis; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry / Q000378:metabolism*; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D011108:Polymers; C009916:nylon 6; D002209:Caprolactam; D043182:Carboxylesterase; D000581:Amidohydrolases; C026250:6-aminohexanoate-dimer hydrolase; D001618:beta-Lactamases