title: X-ray crystallographic analysis of 6-aminohexanoate-dimer hydrolase: molecular basis for the birth of a nylon oligomer-degrading enzyme.
abstract: 6-Aminohexanoate-dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII') that has only approximately 0.5% of the specific activity toward the 6-aminohexanoate-linear dimer, are encoded on plasmid pOAD2 of Arthrobacter sp. (formerly Flavobacterium sp.) KI72. Here, we report the three-dimensional structure of Hyb-24 (a hybrid between the EII and EII' proteins; EII'-level activity) by x-ray crystallography at 1.8 A resolution and refined to an R-factor and R-free of 18.5 and 20.3%, respectively. The fold adopted by the 392-amino acid polypeptide generated a two-domain structure that is similar to the folds of the penicillin-recognizing family of serine-reactive hydrolases, especially to those of d-alanyl-d-alanine-carboxypeptidase from Streptomyces and carboxylesterase from Burkholderia. Enzyme assay using purified enzymes revealed that EII and Hyb-24 possess hydrolytic activity for carboxyl esters with short acyl chains but no detectable activity for d-alanyl-d-alanine. In addition, on the basis of the spatial location and role of amino acid residues constituting the active sites of the nylon oligomer hydrolase, carboxylesterase, d-alanyl-d-alanine-peptidase, and beta-lactamases, we conclude that the nylon oligomer hydrolase utilizes nucleophilic Ser(112) as a common active site both for nylon oligomer-hydrolytic and esterolytic activities. However, it requires at least two additional amino acid residues (Asp(181) and Asn(266)) specific for nylon oligomer-hydrolytic activity. Here, we propose that amino acid replacements in the catalytic cleft of a preexisting esterase with the beta-lactamase fold resulted in the evolution of the nylon oligomer hydrolase.
authors.affiliation: Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, Himeji, Japan. negoro@eng.u-hyogo.ac.jp
authors.forename: Seiji
authors.initials: S
authors.lastname: Negoro
authors.forename: Taku
authors.initials: T
authors.lastname: Ohki
authors.forename: Naoki
authors.initials: N
authors.lastname: Shibata
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Mizuno
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Wakitani
authors.forename: Junya
authors.initials: J
authors.lastname: Tsurukame
authors.forename: Keiji
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Ichitaro
authors.initials: I
authors.lastname: Kawamoto
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takeo
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Higuchi
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000614:Aminocaproates; D015119:Aminocaproic Acid / Q000737:chemistry / Q000378:metabolism; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001673:Biodegradation, Environmental; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000235:genetics; D019143:Evolution, Molecular; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: C026243:6-aminohexanoic acid cyclic dimer; D000614:Aminocaproates; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D000581:Amidohydrolases; C026250:6-aminohexanoate-dimer hydrolase; D015119:Aminocaproic Acid