Molecular design of a nylon-6 byproduct-degrading enzyme from a carboxylesterase with a beta-lactamase fold.
A carboxylesterase with a beta-lactamase fold from Arthrobacter possesses a low level of hydrolytic activity (0.023 mumol.min(-1).mg(-1)) when acting on a 6-aminohexanoate linear dimer byproduct of the nylon-6 industry (Ald). G181D/H266N/D370Y triple mutations in the parental esterase increased the Ald-hydrolytic activity 160-fold. Kinetic studies showed that the triple mutant possesses higher affinity for the substrate Ald (K(m) = 2.0 mm) than the wild-type Ald hydrolase from Arthrobacter (K(m) = 21 mm). In addition, the k(cat)/K(m) of the mutant (1.58 s(-1).mm(-1)) was superior to that of the wild-type enzyme (0.43 s(-1).mm(-1)), demonstrating that the mutant efficiently converts the unnatural amide compounds even at low substrate concentrations, and potentially possesses an advantage for biotechnological applications. X-ray crystallographic analyses of the G181D/H266N/D370Y enzyme and the inactive S112A-mutant-Ald complex revealed that Ald binding induces rotation of Tyr370/His375, movement of the loop region (N167-V177), and flip-flop of Tyr170, resulting in the transition from open to closed forms. From the comparison of the three-dimensional structures of various mutant enzymes and site-directed mutagenesis at positions 266 and 370, we now conclude that Asn266 makes suitable contacts with Ald and improves the electrostatic environment at the N-terminal region of Ald cooperatively with Asp181, and that Tyr370 stabilizes Ald binding by hydrogen-bonding/hydrophobic interactions at the C-terminal region of Ald.
Kawashima
Yasuyuki
Y
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, Japan.
Ohki
Taku
T
Shibata
Naoki
N
Higuchi
Yoshiki
Y
Wakitani
Yoshiaki
Y
Matsuura
Yusuke
Y
Nakata
Yusuke
Y
Takeo
Masahiro
M
Kato
Dai-ichiro
D
Negoro
Seiji
S
D019943:Amino Acid Substitution; D001173:Arthrobacter / Q000201:enzymology; D002209:Caprolactam / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D043182:Carboxylesterase / Q000737:chemistry*; D020134:Catalytic Domain; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D013329:Structure-Activity Relationship; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
D011108:Polymers; C009916:nylon 6; D002209:Caprolactam; D043182:Carboxylesterase; D001618:beta-Lactamases