title: X-ray crystallographic analysis of the 6-aminohexanoate cyclic dimer hydrolase: catalytic mechanism and evolution of an enzyme responsible for nylon-6 byproduct degradation.
abstract: We performed x-ray crystallographic analyses of the 6-aminohexanoate cyclic dimer (Acd) hydrolase (NylA) from Arthrobacter sp., an enzyme responsible for the degradation of the nylon-6 industry byproduct. The fold adopted by the 472-amino acid polypeptide generated a compact mixed alpha/beta fold, typically found in the amidase signature superfamily; this fold was especially similar to the fold of glutamyl-tRNA(Gln) amidotransferase subunit A (z score, 49.4) and malonamidase E2 (z score, 44.8). Irrespective of the high degree of structural similarity to the typical amidase signature superfamily enzymes, the specific activity of NylA for glutamine, malonamide, and indoleacetamide was found to be lower than 0.5% of that for Acd. However, NylA possessed carboxylesterase activity nearly equivalent to the Acd hydrolytic activity. Structural analysis of the inactive complex between the activity-deficient S174A mutant of NylA and Acd, performed at 1.8 A resolution, suggested the following enzyme/substrate interactions: a Ser(174)-cis-Ser(150)-Lys(72) triad constitutes the catalytic center; the backbone N in Ala(171) and Ala(172) are involved in oxyanion stabilization; Cys(316)-S(gamma) forms a hydrogen bond with nitrogen (Acd-N(7)) at the uncleaved amide bond in two equivalent amide bonds of Acd. A single S174A, S150A, or K72A substitution in NylA by site-directed mutagenesis decreased the Acd hydrolytic and esterolytic activities to undetectable levels, indicating that Ser(174)-cis-Ser(150)-Lys(72) is essential for catalysis. In contrast, substitutions at position 316 specifically affected Acd hydrolytic activity, suggesting that Cys(316) is responsible for Acd binding. On the basis of the structure and functional analysis, we discussed the catalytic mechanisms and evolution of NylA in comparison with other Ser-reactive hydrolases.
authors.affiliation: Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, Hyogo 671-2201, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Yasuhira
authors.forename: Naoki
authors.initials: N
authors.lastname: Shibata
authors.forename: Go
authors.initials: G
authors.lastname: Mongami
authors.forename: Yuki
authors.initials: Y
authors.lastname: Uedo
authors.forename: Yu
authors.initials: Y
authors.lastname: Atsumi
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Kawashima
authors.forename: Atsushi
authors.initials: A
authors.lastname: Hibino
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Lee
authors.forename: Dai-ichiro
authors.initials: D
authors.lastname: Kato
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takeo
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Higuchi
authors.forename: Seiji
authors.initials: S
authors.lastname: Negoro
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001673:Biodegradation, Environmental; D002209:Caprolactam / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D020125:Mutation, Missense; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000502:physiology; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D011108:Polymers; C009916:nylon 6; D002209:Caprolactam; D000581:Amidohydrolases; C019683:6-aminohexanoate-cyclic-dimer hydrolase