Molecular design of a nylon‐6 byproduct‐degrading enzyme from a carboxylesterase with a β‐lactamase fold

Abstract

A carboxylesterase with a β‐lactamase fold from Arthrobacter possesses a low level of hydrolytic activity (0.023 μmol·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 (Km = 2.0 mm) than the wild‐type Ald hydrolase from Arthrobacter (Km = 21 mm). In addition, the kcat/Km 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.

X-ray Crystallographic Analysis of the 6-Aminohexanoate Cyclic Dimer Hydrolase

“…This model is consistent with our finding that Ald hydrolytic activity is significantly affected by amino acid substitutions at positions 170, 181, 187, 264, 266, and 370, responsible for Ald binding, whereas amino acid substitutions at these positions barely affect the esterase activity (37)(38)(39)(40)(41). A molecular dynamic simulation for NylB/NylBЈ has suggested that the substrateunbound open form is maintained at an energy minimum and requires activation energy to transition to the substrate-bound closed form (38).…”

“…We have speculated that the release of 6-aminohexanoate (corresponding to the C-terminal half of Ald) triggers the penetration of water molecules to the catalytic center (38). On the basis of these results, we have proposed that NylB has evolved from a pre-existing esterase with a ␤-lactamase fold (37)(38)(39)(40)(41). Present x-ray crystallographic analysis revealed that NylA is classified as a member of the AS superfamily and utilizes the distinct catalytic triad Ser-cis-Ser-Lys.…”

“…Another catalytic triad, Ser-Tyr-Lys, has been identified in NylB (Ald-hydrolase) (37)(38)(39)(40)(41), NylBЈ carboxylesterase (37-41), EstB carboxylesterase (42), DD-peptidase (43), and class C ␤-lactamase (44). NylB and a NylB/NylBЈ hybrid protein (Hyb24DN; having a NylB-level of Ald hydrolytic activity) effectively recognize substrates having 6-aminohexanoate (nylon unit) as N-terminal residues in the substrate but possess activities for various carboxylesters (37)(38)(39)(40)(41).…”

Crystallization and X-ray diffraction analysis of nylon hydrolase (NylC) fromArthrobactersp. KI72

“…We have studied the degradation of polymeric nylon-6 and the byproducts of the manufacture of nylon-6, namely 6-aminohexanoate oligomers (nylon oligomers), as a model system (Okada et al, 1983;Negoro, 2000). We found that three enzymes, 6-aminohexanoatecyclic dimer hydrolase (NylA; Yasuhira et al, 2010), 6-aminohexanoate-dimer hydrolase (NylB; Negoro et al, 2005;Ohki et al, 2006Ohki et al, , 2009Kawashima et al, 2009) and nylon hydrolase (NylC), are responsible for the degradation of nylon-6 and its related compounds.…”

Hydration effects on enzyme–substrate complex of nylon oligomer hydrolase: inter-fragment interaction energy study by the fragment molecular orbital method

“…The initial atomic coordinates of NylB were obtained from the X-ray crystallographic experimental structure of PDB ID 2ZMA taken from Protein Data Bank [18], consisting of 392 amino acid residues with a total charge of 15 e. To model the wild-type structure, Ala112 was mutated to serine, and the missing hydrogen atoms and amino acid residues in the PDB file were added by Amber Tool [19]. The unknown parameters for the substrate, 6-aminohexanoate linear dimer (ALD), were calculated by the G03 [20] and Leap programs with a General AMBER force field [21].…”