Publications

• Mutational analysis of 6-aminohexanoate-dimer hydrolase: relationship between nylon oligomer hydrolytic and esterolytic activities.

  Ohki T., Wakitani Y., Takeo M., Yasuhira K., Shibata N., Higuchi Y., Negoro S.

  Carboxylesterase (EII') from Arthrobacter sp. KI72 has 88% homology to 6-aminohexanoate-dimer hydrolase (EII) and possesses ca. 0.5% of the level of 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity of EII. To study relationship between Ald-hydrolytic and esterolytic activities, random mutat ... >> More

  Carboxylesterase (EII') from Arthrobacter sp. KI72 has 88% homology to 6-aminohexanoate-dimer hydrolase (EII) and possesses ca. 0.5% of the level of 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity of EII. To study relationship between Ald-hydrolytic and esterolytic activities, random mutations were introduced into the gene for Hyb-24 (an EII/EII' hybrid with the majority of the sequence deriving for EII' and possessing an EII'-like level of Ald-hydrolytic activity). Either a G181D or a D370Y substitution in Hyb-24 increased the Ald-hydrolytic activity ca. 10-fold, and a G181D/D370Y double substitution increased activity ca. 100-fold. On the basis of kinetic studies and the three-dimensional structure of the enzyme, we suggest that binding of Ald is improved by these mutations. D370Y increased esterolytic activity for glycerylbutyrate ca. 30-50-fold, whereas G181D decreased the activity to 30% of the parental enzyme. << Less

  FEBS Lett 580:5054-5058(2006) [PubMed] [EuropePMC]

• Crystallization and X-ray diffraction analysis of 6-aminohexanoate-dimer hydrolase from Arthrobacter sp. KI72.

  Ohki T., Mizuno N., Shibata N., Takeo M., Negoro S., Higuchi Y.

  To investigate the structure-function relationship between 6-aminohexanoate-dimer hydrolase (EII) from Arthrobacter sp. and a cryptic protein (EII') which shows 88% sequence identity to EII, a hybrid protein (named Hyb-24) of EII and EII' was overexpressed, purified and crystallized using the sitt ... >> More

  To investigate the structure-function relationship between 6-aminohexanoate-dimer hydrolase (EII) from Arthrobacter sp. and a cryptic protein (EII') which shows 88% sequence identity to EII, a hybrid protein (named Hyb-24) of EII and EII' was overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant in MES buffer pH 6.5. The crystal belongs to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 96.37, c = 113.09 A. Diffraction data were collected from native and methylmercuric chloride derivative crystals to resolutions of 1.75 and 1.80 A, respectively. << Less

  Acta Crystallogr Sect F Struct Biol Cryst Commun 61:928-930(2005) [PubMed] [EuropePMC]

• X-ray crystallographic analysis of 6-aminohexanoate-dimer hydrolase: molecular basis for the birth of a nylon oligomer-degrading enzyme.

  Negoro S., Ohki T., Shibata N., Mizuno N., Wakitani Y., Tsurukame J., Matsumoto K., Kawamoto I., Takeo M., Higuchi Y.

  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 Fl ... >> More

  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. << Less

  J Biol Chem 280:39644-39652(2005) [PubMed] [EuropePMC]

• Purification and characterization of 6-aminohexanoic-acid-oligomer hydrolase of Flavobacterium sp. Ki72.

  Kinoshita S., Terada T., Taniguchi T., Takene Y., Masuda S., Matsunaga N., Okada H.

  6-Aminohexanoic-oligomer hydrolase of Flavobacterium sp. KI72 was purified to homogeneity by column chromatography three times, and by preparation polyacrylamide gel electrophoresis twice. The purified enzyme had the following characteristics. 1. The molecular weight was estimated to be 84000 by S ... >> More

  6-Aminohexanoic-oligomer hydrolase of Flavobacterium sp. KI72 was purified to homogeneity by column chromatography three times, and by preparation polyacrylamide gel electrophoresis twice. The purified enzyme had the following characteristics. 1. The molecular weight was estimated to be 84000 by Sephadex G-200 molecular-sieve chromatography. The enzyme consisted of two homologous subunits of 42000, judged from sodium dodecylsulfate/polyacrylamide gel electrophoresis. 2. The optimum pH for activity was between 8 and 9, the optimum temperature was 40 degrees C for a 1-h reaction. The Michaelis-Menten constants and turnover numbers for the 6-aminohexanoic acid dimer and trimer were 5.9 mM and 2.4 s-1, and 6.2 mM and 2.0 s-1 respectively. 3. The enzyme was inhibited by 0.37 mM diisopropylfluorophosphate and by 0.013 mM p-chloromercuribenzoate. 4. The enzyme was active on 6-aminohexanoic acid oligomers from dimer to hexamer and icosamer but not on hectamer, and the activity decreased with the increase of the polymerization number of the oligomer. The oligomers were hydrolyzed so as to remove the 6-aminohexanoic acid residue successively from the amino terminus. The enzyme could not hydrolyze other linear amides, cyclic amides, dipeptides, tripeptides or casein. 5. 6-aminohexanoic-acid-oligomer hydrolase was classified as a new member of the linear amidases (EC 3.5.1.-). << Less

  Eur J Biochem 116:547-551(1981) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.