Kengo Yasuhira

X-ray Crystallographic Analysis of the 6-Aminohexanoate Cyclic Dimer Hydrolase CATALYTIC MECHANISM AND EVOLUTION OF AN ENZYME RESPONSIBLE FOR NYLON-6 BYPRODUCT DEGRADATION

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 α/β fold, typically found in the amidase signature superfamily; this fold was especially similar to the fold of glutamyl-tRNAGln 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 Å resolution, suggested the following enzyme/substrate interactions: a Ser174-cis-Ser150-Lys72 triad constitutes the catalytic center; the backbone N in Ala171 and Ala172 are involved in oxyanion stabilization; Cys316-Sγ forms a hydrogen bond with nitrogen (Acd-N7) 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 Ser174-cis-Ser150-Lys72 is essential for catalysis. In contrast, substitutions at position 316 specifically affected Acd hydrolytic activity, suggesting that Cys316 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.

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

ABSTRACT 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.

Genetic Organization of Nylon-Oligomer-Degrading Enzymes from Alkalophilic Bacterium, Agromyces sp. KY5R

A 15-kb gene locus including nylon-oligomer-degrading genes from the chromosome of an alkalophilic bacterium, Agromyces sp. KY5R, was cloned and sequenced. The genetic organization was similar to the DNA region flanked by directly repeated IS6100 sequences on the nylon-oligomer-degradative plasmid pOAD2. However, we found several genetic rearrangements between the two DNA regions. Here, we discuss the possible mechanisms underlying the genetic rearrangements.

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

Background: Biodegradation of polyamides is important from the industrial and environmental point of view. Results: We identified the catalytic residue of nylon hydrolase as Thr-267 and enhanced the protein thermostability by 36 °C (Tm = 88 °C) by introducing mutations at the subunit interfaces of tetramer structure. Conclusion: We revealed the mechanism of nylon-6 hydrolysis. Significance: We established an approach to biodegrade polymeric nylon-6. We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of Tm = 52 °C, enhanced the protein thermostability by 36 °C (Tm = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000–25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500–3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.

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

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.

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

6-Aminohexanoate-cyclic-dimer hydrolase (EI) from Arthrobacter sp. KI72 was expressed in Escherichia coli and purified by anion-exchange chromatography. EI was crystallized by the sitting-drop vapour-diffusion method with sodium citrate as precipitant in imidazole buffer pH 8.0. The crystal is hexagonal, with unit-cell parameters a = b = 130.75, c = 58.23 A. Diffraction data were collected from native and mercury(II) dichloride-derivative crystals to resolutions of 1.90 and 2.06 A, respectively.

Crystallization and X-ray diffraction analysis of nylon-oligomer hydrolase (NylC) from Agromyces sp. KY5R

6-Aminohexanoate-oligomer hydrolase (NylC) from Agromyces sp. KY5R was expressed in Escherichia coli JM109 and purified by ammonium sulfate fractionation, anion-exchange column chromatography and gel-filtration chromatography. NylC was crystallized by the sitting-drop vapour-diffusion method with sodium citrate as a precipitant in 0.1 M HEPES buffer pH 7.5 containing 0.2 M NaCl. Diffraction data were collected from native and K(2)PtCl(4)-derivative crystals to resolutions of 2.00 and 2.20 Å, respectively. The obtained crystal was plate-shaped, with an I-centred orthorhombic space group and unit-cell parameters a = 155.86, b = 214.45, c = 478.80 Å. The anomalous difference Patterson map of the K(2)PtCl(4)-derivative crystal suggested that the space group was I222 rather than I2(1)2(1)2(1).

Crystallization and X-ray diffraction analysis of nylon hydrolase (NylC) from Arthrobacter sp. KI72

Nylon hydrolase (NylC) encoded by Arthrobacter plasmid pOAD2 (NylCp2) was expressed in Escherichia coli JM109 and purified by ammonium sulfate fractionation, anion-exchange column chromatography and gel-filtration chromatography. NylCp2 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 C2221, 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.