Nylon-oligomer Degrading Enzyme/Substrate Complex: Catalytic Mechanism of 6-Aminohexanoate-dimer Hydrolase

Negoro, Seiji; Ohki, T.; Shibata, Naoki; Sasa, Kazuhiro; Hayashi, Hisako; Nakano, Hidehiko; Yasuhira, Kengo; Kato, Dai‐ichiro; Takeo, Masahiro; Higuchi, Yoshiki

doi:10.1016/j.jmb.2007.04.043

Cited by 54 publications

(97 citation statements)

References 45 publications

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“…Another catalytic triad, Ser-Tyr-Lys, has been identified in NylB (Ald-hydrolase) (37)(38)(39)(40)(41), NylBЈ carboxylesterase (37-41), EstB carboxylesterase (42), DD-peptidase (43), and class C ␤-lactamase (44). NylB and a NylB/NylBЈ hybrid protein (Hyb24DN; having a NylB-level of Ald hydrolytic activity) effectively recognize substrates having 6-aminohexanoate (nylon unit) as N-terminal residues in the substrate but possess activities for various carboxylesters (37)(38)(39)(40)(41).…”

Section: Comparison With Other Ser-reactive Hydrolases and Evolutionamentioning

confidence: 99%

X-ray Crystallographic Analysis of the 6-Aminohexanoate Cyclic Dimer Hydrolase

Yasuhira

Shibata

Mongami

et al. 2010

Journal of Biological Chemistry

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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 ␣/␤ 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. Nylon-6 is produced by ring cleavage polymerization of ⑀-caprolactam and consists of more than 100 units of 6-aminohexanoate. During the polymerization reaction, however, some molecules fail to polymerize and remain oligomers, whereas others undergo head-to-tail condensation to form cyclic oligomers (1, 2). These byproducts (designated nylon oligomers) contribute to an increase in industrial waste material into the environment. Therefore, biodegradation of the xenobiotic compounds is important in terms of environmental points of view. In addition, the biological system for degradation provides us with a suitable system to study how microorganisms have evolved specific enzymes responsible for this degradation (1, 2).Previous biochemical studies in a nylon oligomer-degrading bacterium Arthrobacter sp. strain KI72 have revealed that three enzymes, 6-aminohexanoate cyclic dimer hydrolase (NylA), 4 6-aminohexanoate dimer hydrolase (NylB), and endo-type 6-aminohexanoate oligomer hydrolase (NylC), are responsible for the degradation of nylon oligomers (1, 2). NylA specifically hydrolyzes one of the two equivalent amide bonds in Acd, generating a 6-aminohexanoate linear dimer (Ald) (see Fig. 1A). However, NylA is barely active on Ald (substrate for NylB), 6-aminohexanoate cyclic oligomers (degree of polymerization Ͼ 3, substrates for NylC), and more than 60 kinds of various amide compounds, including peptides and ␤-lactams (1-3). NylA is a homodimeric enzyme, and Acd hydrolytic activity is inhibited by diisopropylfluorophosphate, a Ser-enzyme-specific inhibitor, suggesting that Ser is involved in the catalytic function (3). NylA has also been found in Pseudomonas sp. NK87 and six alkalophilic strains, including Kocuria sp. KY2 (4 -6). All of the NylA proteins identified so far have been * This work was supported in part by a grant-in-aid for scientific research from the Japan Society for Promotion of Science and by grants from the Global Centers of Excellence Program, the National Project on Protein Structural and Functional Analyses, the Core Research for Evolutional Science and Technolo...

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Section: Comparison With Other Ser-reactive Hydrolases and Evolutionamentioning

confidence: 99%

X-ray Crystallographic Analysis of the 6-Aminohexanoate Cyclic Dimer Hydrolase

Yasuhira

Shibata

Mongami

et al. 2010

Journal of Biological Chemistry

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“…[see Addendum]) as a model system (9, 10). We found that three enzymes, the Ahx cyclicdimer hydrolase (NylA) (6), the Ahx dimer hydrolase (NylB) (7,12,13), and the Ahx endo-type-oligomer hydrolase (NylC) (4, 11), are responsible for the degradation of the nylon oligomers ( Fig. 1).…”

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confidence: 99%

6-Aminohexanoate Oligomer Hydrolases from the Alkalophilic Bacteria Agromyces sp. Strain KY5R and Kocuria sp. Strain KY2

Yasuhira

Tanaka

Shibata

et al. 2007

Appl Environ Microbiol

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Alkalophilic, nylon oligomer-degrading strains, Agromyces sp. and Kocuria sp., were isolated from the wastewater of a nylon-6 factory and from activated sludge from a sewage disposal plant. The 6-aminohexanoate oligomer hydrolases (NylC) from the alkalophilic strains had 95.8 to 98.6% similarity to the enzyme in neutrophilic Arthrobacter sp. but had superior thermostability, activity under alkaline conditions, and affinity for nylon-related substrates, which would be advantageous for biotechnological applications.The biodegradation of unnatural synthetic compounds which have been released into the natural environment with the development of the chemical industry provides a suitable system for investigating how microorganisms evolve the enzymes essential for the degradation of such xenobiotic compounds. We have studied the degradation of by-products of the manufacture of nylon-6, namely, 6-aminohexanoate (Ahx) oligomers (nylon oligomers), by Arthrobacter sp. strain KI72 (formerly called a Flavobacterium sp. [see Addendum]) as a model system (9, 10). We found that three enzymes, the Ahx cyclicdimer hydrolase (NylA) (6), the Ahx dimer hydrolase (NylB) (7,12,13), and the Ahx endo-type-oligomer hydrolase (NylC) (4, 11), are responsible for the degradation of the nylon oligomers ( Fig. 1).Nylon oligomers are discharged from nylon factories as an alkaline solution. However, the optimum pH for the growth of strain KI72 is approximately 7, and thus far, no alkalophilic, nylon oligomer-degrading strains have been isolated. Therefore, if such microorganisms could be isolated, they would be useful for the direct treatment of wastes from nylon-6 factories. In addition, comparative analyses for the responsible enzymes are expected to provide information on the evolutionary and functional divergence of these enzymes. In this paper, we report the isolation of alkalophilic, nylon oligomer-degrading bacteria, their genetic cloning, and the characterization of their nylon oligomer-degrading enzymes.Isolation of alkalophilic nylon oligomer-degrading bacteria. The nylon oligomer mixture (NOM) (Toyobo Co., Tsuruga, Japan) used is a mixture of Ahx cyclic and linear oligomers. To obtain the cyclic-oligomer-enriched fraction used for bacterial screening, the NOM was extensively washed with hot water on filter paper to remove water-soluble linear oligomers. After being dried, washed NOM (NOM-W) was obtained. Thin-layer chromatography (TLC) analysis revealed that no free amino groups were detected by ninhydrin in NOM-W, which indicates the absence of the linear oligomer, while several spots appeared in the original NOM (Fig. 2C).Microorganisms included in activated sludge from a sewage disposal plant (sample 1) and in wastewater from a nylon-6 factory (sample 2) were enriched in NOM10 medium (0.4% NOM-W, 0.2% Na 3 PO 4 ⅐ 12H 2 O, 0.1% K 2 HPO 4 , 0.26% Na 2 CO 3 , 0.2% NaHCO 3 , 0.02% MgSO 4 ⅐ 7H 2 O, 0.5% NaCl, 0.01% yeast extract, pH 10). The cultures were diluted with sterilized water and plated on LB-NOM10 plates (LB plates containing...

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“…11,12,[18][19][20][21][22] Amino acid alignment of the esterase of A. globiformis with the penicillin-binding proteins and family VIII esterases shows that the consensus active site motif of Ser-X-X-Lys, found exclusively among the homologous enzymes, 11) is also considered to be the active site of the esterase in this study (Ser59-Cys-Ser-Lys62), while the other two elements consist of slightly different amino acids, such as Tyr-His-Ala and Lys-Ser-His respectively. In this study, we focused on identifying the catalytic residue and elucidating the role of the lysine residue one helix turn downstream of the serine, and considered the similarities and differences in catalytic mechanism between the esterase in this study and the homologous enzymes through DFP-labeling and site-directed mutagenesis experiments.…”

Section: Resultsmentioning

confidence: 99%

“…Previous reports have discussed the mechanism of catalytic events, i.e., acylation and deacylation steps, of these penicillin-binding proteins, and have reported that several residues in the conserved elements are responsible for catalytic activity. [18][19][20][21][22] In this study, in order to identify the catalytic residues of esterase from A. globiformis and the differences and similarities in the hydrolytic mechanism between this esterase and the other homologous enzymes, both DFPlabeling and site-directed mutagenesis experiments were conducted. Covalent labeling of the esterase with the inhibitor followed by mass spectrometric analysis was performed to identify the active-site nucleophile.…”

Section: Identification Of the Catalytic Residues Of Carboxylesterasementioning

confidence: 99%

Identification of the Catalytic Residues of Carboxylesterase fromArthrobacter globiformisby Diisopropyl Fluorophosphate-Labeling and Site-Directed Mutagenesis

Nishizawa¹,

Yabusaki

Kanaoka³

2011

Bioscience, Biotechnology, and Biochemistry

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Nylon-oligomer Degrading Enzyme/Substrate Complex: Catalytic Mechanism of 6-Aminohexanoate-dimer Hydrolase

Cited by 54 publications

References 45 publications

X-ray Crystallographic Analysis of the 6-Aminohexanoate Cyclic Dimer Hydrolase

X-ray Crystallographic Analysis of the 6-Aminohexanoate Cyclic Dimer Hydrolase

6-Aminohexanoate Oligomer Hydrolases from the Alkalophilic Bacteria Agromyces sp. Strain KY5R and Kocuria sp. Strain KY2

Identification of the Catalytic Residues of Carboxylesterase fromArthrobacter globiformisby Diisopropyl Fluorophosphate-Labeling and Site-Directed Mutagenesis

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