Three-dimensional Structure of Nylon Hydrolase and Mechanism of Nylon-6 Hydrolysis

Negoro, Seiji; Shibata, Naoki; Tanaka, Yusuke; Yasuhira, Kengo; Shibata, Hiroshi; Hashimoto, Haruka; Lee, Young-Ho; Oshima, Shohei; Santa, Ryuji; Mochiji, Kōzō; Goto, Yuji; Ikegami, Takahisa; Nagai, Keisuke; Kato, Dai‐ichiro; Takeo, Masahiro; Higuchi, Yoshiki

doi:10.1074/jbc.m111.321992

Cited by 54 publications

(73 citation statements)

References 44 publications

(42 reference statements)

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“…NylC is a member of the N-terminal nucleophile hydrolase superfamily and degrades cyclic and linear oligomers of 6-aminohexanoate with a degree of polymerization greater than three by an endo-type mode (Negoro et al, 1992(Negoro et al, , 2012Kakudo et al, 1993Kakudo et al, , 1995Yasuhira, Uedo et al, 2007). NylC has been found in Arthrobacter sp.…”

Section: Introductionmentioning

confidence: 99%

“…However, the obtained crystal contained 15 monomer molecules in a single asymmetric unit of space group I222, with unit-cell parameters a = 155.86, b = 214.45, c = 478.80 Å . Based on the structural analyses, the 15 monomer molecules constitute three tetramers, one dimer forming a tetramer that is related by a crystallographic twofold axis and one monomer forming a tetramer that is related by crystallographic 222 symmetry in a single asymmetric unit of NylC A (Negoro et al, 2012). In addition, most mutations which significantly affect protein stability among the NylC p2 , NylC A and NylC K proteins were found to be located at the subunit interfaces generating the dimeric or tetrameric structures (Negoro et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

“…Recently, we reported the crystallization conditions and the structural analysis of NylC A at 2.0 Å resolution (Yasuhira et al, 2011;Negoro et al, 2012). However, the obtained crystal contained 15 monomer molecules in a single asymmetric unit of space group I222, with unit-cell parameters a = 155.86, b = 214.45, c = 478.80 Å .…”

Section: Introductionmentioning

confidence: 99%

“…Interestingly, replacement of the NylC p2 sequence with the NylC A /NylC K sequences drastically alters the thermostability of the enzyme. In particular, two mutations (D122G/H130Y) from NylC A and two further mutations (D36A/ E263Q) from NylC K in NylC p2 (heat denaturation temperature T m = 325 K) enhanced the protein thermostability by 36 K (T m = 361 K; Negoro et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

“…Based on the structural analyses, the 15 monomer molecules constitute three tetramers, one dimer forming a tetramer that is related by a crystallographic twofold axis and one monomer forming a tetramer that is related by crystallographic 222 symmetry in a single asymmetric unit of NylC A (Negoro et al, 2012). In addition, most mutations which significantly affect protein stability among the NylC p2 , NylC A and NylC K proteins were found to be located at the subunit interfaces generating the dimeric or tetrameric structures (Negoro et al, 2012). Since crystals containing such a high number of molecules with heterogeneous oligomeric states in the asymmetric unit are inappropriate for further crystallographic analysis of the mutants, we newly screened the crystallization conditions of the NylC p2 protein to obtain a crystal with fewer molecules in an asymmetric unit.…”

Section: Introductionmentioning

confidence: 99%

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Crystallization and X-ray diffraction analysis of nylon hydrolase (NylC) fromArthrobactersp. KI72

et al. 2013

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Nylon hydrolase (NylC) encoded by Arthrobacter plasmid pOAD2 (NylC p2 ) was expressed in Escherichia coli JM109 and purified by ammonium sulfate fractionation, anion-exchange column chromatography and gel-filtration chromatography. NylC p2 was crystallized by the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant in 0.1 M HEPES buffer pH 7.5 containing 0.2 M NaCl and 25% glycerol. Diffraction data were collected from the native crystal to a resolution of 1.60 Å . The obtained crystal was spindle shaped and belonged to the C-centred orthorhombic space group C222 1 , with unit-cell parameters a = 70.84, b = 144.90, c = 129.05 Å . A rotation and translation search gave one clear solution containing two molecules per asymmetric unit.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Crystallization and X-ray diffraction analysis of nylon hydrolase (NylC) fromArthrobactersp. KI72

et al. 2013

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Mutations affecting the internal equilibrium of the reaction catalyzed by 6‐aminohexanoate‐dimer hydrolase

et al. 2016

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The enzyme 6-aminohexanoate-dimer hydrolase catalyzes amide synthesis. The yield of this reverse reaction in 90% t-butyl alcohol was found to vary drastically when enzyme mutants with substitutions of several amino acids located at the entrance of the catalytic cleft were used. Movement of the loop region and the flip-flop of Tyr170 generate a local hydrophobic environment at the catalytic center of the enzyme. Here, we propose that the shift of the internal equilibrium between the enzyme-substrate complex and enzyme-product complex by the 'water-excluding effect' alters the rate of the forward and reverse reactions. Moreover, we suggest that the local hydrophobic environment potentially provides a reaction center suitable for efficient amide synthesis. Keywords: 6-aminohexanoate-dimer hydrolase; amide synthesis; inducedfit mechanism; internal equilibrium; x-ray crystallography Abbreviations Ahx, 6-aminohexanoate; Ald, 6-aminohexanoate-linear dimer; Hyb-24, a NylB/NylB 0 hybrid protein constructed from conserved PvuII sites located 24-amino acid residues downstream of the initiation codons (NylB 0 having T3A/P4R/T5S/S8Q/D15G substitutions); Hyb-24DN, Hyb-24 having G181D/H266N substitutions; Hyb-24DNY, Hyb-24DN having D370Y substitution; Hyb-S4M94, Hyb-24 having A61V/A124V/R187S/ F264C/G291R/G338A/D370Y substitutions; NylB, 6-aminohexaoate-dimer hydrolase; NylB 0 , a carboxylesterase with 88% homology to NylB encoded on plasmid pOAD2.

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Bacterial β‐Aminopeptidases: Structural Insights and Applications for Biocatalysis

Heck

Geueke

Kohler

2012

Chemistry & Biodiversity

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β-Aminopeptidases comprise a class of enzymes with functional and structural similarities. All members of the β-aminopeptidases described to date were isolated from bacterial sources. Uniquely, they catalyze the hydrolysis of β(3) - and/or β(2) -amino acid residues from amides and peptides that are otherwise considered proteolytically stable. Due to this unusual reactivity with β-peptide substrates, β-aminopeptidases have potential to be used as biocatalysts for β-peptide synthesis and for the resolution of enantiomerically pure β-amino acids from racemic substrate mixtures. β-Aminopeptidases are formed from an inactive precursor by posttranslational autoproteolytic cleavage, exposing the catalytic nucleophile at the N-terminus of the newly formed β-polypeptide chain. Such an activation step is a characteristic trait of enzymes of the N-terminal nucleophile (Ntn) hydrolase superfamily. However, classical Ntn hydrolases and β-aminopeptidases differ by the fold of their catalytic cores and are hence likely to originate from distinct evolutionary ancestors. In this contribution, we review the existing literature on β-aminopeptidases, including biochemical and functional studies, as well as structural investigations that recently allowed insights into the catalytic mechanisms of precursor processing and β-peptide conversion.

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Three-dimensional Structure of Nylon Hydrolase and Mechanism of Nylon-6 Hydrolysis

Cited by 54 publications

References 44 publications

Crystallization and X-ray diffraction analysis of nylon hydrolase (NylC) fromArthrobactersp. KI72

Crystallization and X-ray diffraction analysis of nylon hydrolase (NylC) fromArthrobactersp. KI72

Mutations affecting the internal equilibrium of the reaction catalyzed by 6‐aminohexanoate‐dimer hydrolase

Bacterial β‐Aminopeptidases: Structural Insights and Applications for Biocatalysis

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