2ZMA: Crystal Structure Of 6-aminohexanoate-dimer Hydrolase S112a/g181d/h266n/d370y Mutant With Substrate

Citation:
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.
PDB ID: 2ZMADownload
MMDB ID: 70652
PDB Deposition Date: 2008/4/14
Updated in MMDB: 2017/08
Experimental Method:
x-ray diffraction
Resolution: 1.51  Å
Source Organism:
Similar Structures:
Biological Unit for 2ZMA: monomeric; determined by author
Molecular Components in 2ZMA
Label Count Molecule
Protein (1 molecule)
1
6-aminohexanoate-dimer Hydrolase
Molecule annotation
Chemicals (13 molecules)
1
5
2
2
3
1
4
5
* Click molecule labels to explore molecular sequence information.

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