2ZMA

Crystal Structure of 6-Aminohexanoate-dimer Hydrolase S112A/G181D/H266N/D370Y Mutant with Substrate

Summary for 2ZMA

• Entry DOI: 10.2210/pdb2zma/pdb
• Related: 1WYB 1WYC 2DCF 2E8I 2ZM7 2ZM8 2ZM9
• Descriptor: 6-aminohexanoate-dimer hydrolase, SULFATE ION, 6-AMINOHEXANOIC ACID, ... (6 entities in total)
• Functional Keywords: hydrolase, nylon degradation
• Biological source: Flavobacterium sp.; More
• Total number of polymer chains: 1
• Total formula weight: 44303.94

Authors

Ohki, T.,Shibata, N.,Higuchi, Y.,Takeo, M.,Negoro, S. (deposition date: 2008-04-14, release date: 2009-04-07, Last modification date: 2023-11-15)

Primary citation

Kawashima, Y.,Ohki, T.,Shibata, N.,Higuchi, Y.,Wakitani, Y.,Matsuura, Y.,Nakata, Y.,Takeo, M.,Kato, D.,Negoro, S.
Molecular design of a nylon-6 byproduct-degrading enzyme from a carboxylesterase with a beta-lactamase fold
Febs J., 276:2547-2556, 2009

PubMed Abstract: 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.

PubMed: 19476493
DOI: 10.1111/j.1742-4658.2009.06978.x

Experimental method

X-RAY DIFFRACTION (1.51 Å)