Dai-ichiro Kato

Quantum Yields and Kinetics of the Firefly Bioluminescence Reaction of Beetle Luciferases

Quantum yields of firefly bioluminescence reactions were determined for beetle luciferases from the three main families of luminous beetles emitting different bioluminescence colors. Quantum yield (QY) was significantly correlated with luminescence spectrum. The green light‐emitting luciferase of the Brazilian click beetle, Pyrearinus termitilluminans, whose luminescence spectrum had the shortest peak wavelength of all the luciferases investigated, had the highest QY (0.61). Mutant analyses of active site‐substituted Pyrocoelia miyako luciferases showed that, although kcat was decreased by the mutations, the QY was not significantly affected.

Mechanism of 4-Nitrophenol Oxidation in Rhodococcus sp. Strain PN1: Characterization of the Two-Component 4-Nitrophenol Hydroxylase and Regulation of Its Expression

4-Nitrophenol (4-NP) is a toxic product of the hydrolysis of organophosphorus pesticides such as parathion in soil. Rhodococcus sp. strain PN1 degrades 4-NP via 4-nitrocatechol (4-NC) for use as the sole carbon, nitrogen, and energy source. A 5-kb EcoRI DNA fragment previously cloned from PN1 contained a gene cluster (nphRA1A2) involved in 4-NP oxidation. From sequence analysis, this gene cluster is expected to encode an AraC/XylS family regulatory protein (NphR) and a two-component 4-NP hydroxylase (NphA1 and NphA2). A transcriptional assay in a Rhodococcus strain revealed that the transcription of nphA1 is induced by only 4-NP (of several phenolic compounds tested) in the presence of nphR, which is constitutively expressed. Disruption of nphR abolished transcriptional activity, suggesting that nphR encodes a positive regulatory protein. The two proteins of the 4-NP hydroxylase, NphA1 and NphA2, were independently expressed in Escherichia coli and purified by ion-exchange chromatography or affinity chromatography. The purified NphA2 reduced flavin adenine dinucleotide (FAD) with the concomitant oxidation of NADH, while the purified NphA1 oxidized 4-NP into 4-NC almost quantitatively in the presence of FAD, NADH, and NphA2. This functional analysis, in addition to the sequence analysis, revealed that this enzyme system belongs to the two-component flavin-diffusible monooxygenase family. The 4-NP hydroxylase showed comparable oxidation activities for phenol and 4-chlorophenol to that for 4-NP and weaker activities for 3-NP and 4-NC.

Identification and characterization of another 4-nitrophenol degradation gene cluster, nps, in Rhodococcus sp. strain PN1.

4-Nitrophenol (4-NP) is a toxic compound formed in soil by the hydrolysis of organophosphorous pesticides, such as parathion. We previously reported the presence of the 4-NP degradation gene cluster (nphRA1A2) in Rhodococcus sp. strain PN1, which encodes a two-component 4-NP hydroxylase system that oxidizes 4-NP into 4-nitrocatechol. In the current study, another gene cluster (npsC and npsRA2A1B) encoding a similar 4-NP hydroxylase system was cloned from strain PN1. The enzymes from this 4-NP hydroxylase system (NpsA1 and NpsA2) were purified as histidine-tagged (His-) proteins and then characterized. His-NpsA2 showed NADH/FAD oxidoreductase activity, and His-NpsA1 showed 4-NP oxidizing activity in the presence of His-NpsA2. In the 4-NP oxidation using the reconstituted enzyme system (His-NpsA1 and His-NpsA2), hydroquinone (35% of 4-NP disappeared) and hydroxyquinol (59% of 4-NP disappeared) were detected in the presence of ascorbic acid as a reducing reagent, suggesting that, without the reducing reagent, 4-NP was converted into their oxidized forms, 1,4-benzoquinone and 2-hydroxy-1,4-benzoquinone. In addition, in the cell extract of recombinant Escherichia coli expressing npsB, a typical spectral change showing conversion of hydroxyquinol into maleylacetate was observed. These results indicate that this nps gene cluster, in addition to the nph gene cluster, is also involved in 4-NP degradation in strain PN1.

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.

Two alternative modes for optimizing nylon-6 byproduct hydrolytic activity from a carboxylesterase with a β-lactamase fold: X-ray crystallographic analysis of directly evolved 6-aminohexanoate-dimer hydrolase

Promiscuous 6‐aminohexanoate‐linear dimer (Ald)‐hydrolytic activity originally obtained in a carboxylesterase with a β‐lactamase fold was enhanced about 80‐fold by directed evolution using error‐prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb‐S4M94) demonstrated that the enzyme had acquired an increased affinity (Km = 15 mM) and turnover (kcat = 3.1 s−1) for Ald, and that a catalytic center suitable for nylon‐6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb‐S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen‐bonding/hydrophobic interactions between the AldCOOH and Tyr370, a hydrogen‐bonding network from Ser187 to AldNH  3+ , and interaction between AldNH  3+ and Gln27‐Oε derived from another subunit in the homo‐dimeric structure. In wild‐type Ald‐hydrolase (NylB), Ald‐hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with AldNH  3+ (Kawashima et al., FEBS J 2009; 276:2547–2556). We propose here that there exist at least two alternative modes for optimizing the Ald‐hydrolytic activity of a carboxylesterase with a β‐lactamase fold.

Molecular design of a nylon‐6 byproduct‐degrading enzyme from a carboxylesterase with a β‐lactamase fold

A carboxylesterase with a β‐lactamase fold from Arthrobacter possesses a low level of hydrolytic activity (0.023 μmol·min−1·mg−1) when acting on a 6‐aminohexanoate linear dimer byproduct of the nylon‐6 industry (Ald). G181D/H266N/D370Y triple mutations in the parental esterase increased the Ald‐hydrolytic activity 160‐fold. Kinetic studies showed that the triple mutant possesses higher affinity for the substrate Ald (Km = 2.0 mm) than the wild‐type Ald hydrolase from Arthrobacter (Km = 21 mm). In addition, the kcat/Km of the mutant (1.58 s−1·mm−1) was superior to that of the wild‐type enzyme (0.43 s−1·mm−1), demonstrating that the mutant efficiently converts the unnatural amide compounds even at low substrate concentrations, and potentially possesses an advantage for biotechnological applications. X‐ray crystallographic analyses of the G181D/H266N/D370Y enzyme and the inactive S112A‐mutant–Ald complex revealed that Ald binding induces rotation of Tyr370/His375, movement of the loop region (N167–V177), and flip‐flop of Tyr170, resulting in the transition from open to closed forms. From the comparison of the three‐dimensional structures of various mutant enzymes and site‐directed mutagenesis at positions 266 and 370, we now conclude that Asn266 makes suitable contacts with Ald and improves the electrostatic environment at the N‐terminal region of Ald cooperatively with Asp181, and that Tyr370 stabilizes Ald binding by hydrogen‐bonding/hydrophobic interactions at the C‐terminal region of Ald.

Function of a Glutamine Synthetase-Like Protein in Bacterial Aniline Oxidation via γ-Glutamylanilide

Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl2. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell.