2ZM9

Structure of 6-Aminohexanoate-dimer Hydrolase, A61V/S112A/A124V/R187S/F264C/G291R/G338A/D370Y mutant (Hyb-S4M94) with Substrate

Summary for 2ZM9

• Entry DOI: 10.2210/pdb2zm9/pdb
• Related: 1WYB 1WYC 2DCF 2E8I 2ZM7 2ZM8 2ZMA
• Descriptor: 6-aminohexanoate-dimer hydrolase, SULFATE ION, 6-AMINOHEXANOIC ACID, ... (6 entities in total)
• Functional Keywords: alpha-beta, hydrolase, nylon degradation
• Biological source: Flavobacterium sp.; More
• Total number of polymer chains: 1
• Total formula weight: 43960.82

Authors

Ohki, T.,Shibata, N.,Higuchi, Y.,Kawashima, Y.,Takeo, M.,Kato, D.,Negoro, S. (deposition date: 2008-04-14, release date: 2009-04-14, Last modification date: 2023-11-15)

Primary citation

Ohki, T.,Shibata, N.,Higuchi, Y.,Kawashima, Y.,Takeo, M.,Kato, D.,Negoro, S.
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.
Protein Sci., 18:1662-1673, 2009

PubMed 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.

PubMed: 19521995
DOI: 10.1002/pro.185

Experimental method

X-RAY DIFFRACTION (1.5 Å)