7YU1

Structure of 6-aminohexanoate-oligomer hydrolase NylC precursor, D122G/H130Y/T267C mutant

Summary for 7YU1

• Entry DOI: 10.2210/pdb7yu1/pdb
• Descriptor: 6-aminohexanoate-oligomer endohydrolase, GLYCEROL, SODIUM ION, ... (5 entities in total)
• Functional Keywords: nylon oligomer, hydrolase
• Biological source: Arthrobacter
• Total number of polymer chains: 2
• Total formula weight: 74726.26

Authors

Negoro, S.,Higuchi, Y. (deposition date: 2022-08-16, release date: 2023-03-01, Last modification date: 2023-11-29)

Primary citation

Negoro, S.,Shibata, N.,Kato, D.I.,Tanaka, Y.,Yasuhira, K.,Nagai, K.,Oshima, S.,Furuno, Y.,Yokoyama, R.,Miyazaki, K.,Takeo, M.,Hengphasatporn, K.,Shigeta, Y.,Lee, Y.H.,Higuchi, Y.
X-ray crystallographic and mutational analysis of the NylC precursor: catalytic mechanism of autocleavage and substrate hydrolysis of nylon hydrolase.
Febs J., 290:3400-3421, 2023

PubMed Abstract: Nylon hydrolase (NylC), a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, is responsible for the degradation of various aliphatic nylons, including nylon-6 and nylon-66. NylC is initially expressed as an inactive precursor (36 kDa), but the precursor is autocatalytically cleaved at Asn266/Thr267 to generate an active enzyme composed of 27 and 9 kDa subunits. We isolated various mutants with amino acid changes at the catalytic centre. X-ray crystallographic analysis revealed that the NylC precursor forms a doughnut-shaped quaternary structure composed of four monomers (molecules A-D) with D2 symmetry. Catalytic residues in the precursor are covered by loop regions at the A/B interface (equivalent to the C/D interface). However, the catalytic residues are exposed to the solvent environment through autocleavage followed by movements of the loop regions. T267A, D306A and D308A mutations resulted in a complete loss of autocleavage. By contrast, in the T267S mutant, autocleavage proceeded slowly at a constant reaction rate (k = 2.8 × 10  s ) until complete conversion, but the reaction was inhibited by K189A and N219A mutations. Based on the crystallographic and molecular dynamic simulation analyses, we concluded that the Asp308-Asp306-Thr267 triad, resembling the Glu-Ser-Ser triad conserved in Ntn-hydrolase family enzymes, is responsible for autocleavage and that hydrogen-bonding networks connecting Thr267 with Lys189 and Asn219 are required for increasing the nucleophilicity of Thr267-OH in both the water accessible and water inaccessible systems. Furthermore, we determined that NylC employs the Asp308-Asp306-Thr267 triad as catalytic residues for substrate hydrolysis, but the reaction requires Lys189 and Tyr146 as additional catalytic/substrate-binding residues specific for nylon hydrolysis.

PubMed: 36799721
DOI: 10.1111/febs.16755

Experimental method

X-RAY DIFFRACTION (1.13 Å)