根来 誠司

3-Methyl-4-nitrophenol (3M4NP) is formed in soil as a hydrolysis product of fenitrothion, one of the major organophosphorus pesticides. A Pseudomonas strain was isolated as a 3M4NP degrader from a crop soil and designated TSN1. This strain utilized 3M4NP as a sole carbon and energy source. To elucidate the biodegradation pathway, we performed transposon mutagenesis with pCro2a (mini-Tn5495) and obtained three mutants accumulating a dark pink compound(s) from 3M4NP. Rescue cloning and sequence analysis revealed that in all mutants, the transposon disrupted an identical aromatic compound meta-cleaving dioxygenase gene, and a monooxygenase gene was located just downstream of the dioxygenase gene. These two genes were designated mnpC and mnpB, respectively. The gene products showed high identity with the methylhydroquinone (MHQ) monooxygenase (58%) and the 3-methylcatechol 2,3-dioxygenase (54%) of a different 3M4NP degrader Burkholderia sp. NF100. The transposon mutants converted 3M4NP or MHQ into two identical metabolites, one of which was identified as 2-hydroxy-5-methyl-1,4-benzoquinone (2H5MBQ) by GC/MS analysis. Furthermore, two additional genes (named mnpAl and mnpA2), almost identical to the p-nitrophenol monooxygenase and the p-benzoquinone reductase genes of Pseudomonas sp. WBC-3, were isolated from the total DNA of strain TSN1. Disruption of mnpAl resulted in the complete loss of the 3M4NP degradation activity, demonstrating that mnpA1 encodes the initial monooxygenase for 3M4NP degradation. The purified mnpA2 gene product could efficiently reduce methyl p-benzoquinone (MBQ) into MHQ. These results suggest that strain TSN1 degrades 3M4NP via MBQ, MHQ, and 2H5MBQ in combination with mnpA1A2 and mnpCB, existing at different loci on the genome. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.

Nylon hydrolase (NylC) is initially expressed as an inactive precursor (36 kDa). The precursor is cleaved autocatalytically at Asn266/Thr267 to generate an active enzyme composed of an a subunit (27 kDa) and a beta subunit (9 kDa). Four alpha beta heterodimers (molecules A-D) form a doughnut-shaped quaternary structure. In this study, the thermostability of the parental NylC was altered by amino acid substitutions located at the A/D interface (D122G/H130Y/D36A/L137A) or the A/B interface (E263Q) and spanned a range of 47 degrees C. Considering structural, biophysical, and biochemical analyses, we discuss the structural basis of the stability of nylon hydrolase. From the analytical centrifugation data obtained regarding the various mutant enzymes, we conclude that the assembly of the monomeric units is dynamically altered by the mutations. Finally, we propose a model that can predict whether the fate of the nascent polypeptide will be correct subunit assembly, inappropriate protein-protein interactions causing aggregation, or intracellular degradation of the polypeptide.

Arthrobacter sp. strain KI72 grows on a 6-aminohexanoate oligomer, which is a by-product of nylon-6 manufacturing, as a sole source of carbon and nitrogen. We cloned the two genes, nylD (1) and nylE (1) , responsible for 6-aminohexanoate metabolism on the basis of the draft genomic DNA sequence of strain KI72. We amplified the DNA fragments that encode these genes by polymerase chain reaction using a synthetic primer DNA homologous to the 4-aminobutyrate metabolic enzymes. We inserted the amplified DNA fragments into the expression vector pColdI in Escherichia coli, purified the His-tagged enzymes to homogeneity, and performed biochemical studies. We confirmed that 6-aminohexanoate aminotransferase (NylD(1)) catalyzes the reaction of 6-aminohexanoate to adipate semialdehyde using alpha-ketoglutarate, pyruvate, and glyoxylate as amino acceptors, generating glutamate, alanine, and glycine, respectively. The reaction requires pyridoxal phosphate (PLP) as a cofactor. For further metabolism, adipate semialdehyde dehydrogenase (NylE(1)) catalyzes the oxidative reaction of adipate semialdehyde to adipate using NADP(+) as a cofactor. Phylogenic analysis revealed that NylD(1) should be placed in a branch of the PLP-dependent aminotransferase sub III, while NylE(1) should be in a branch of the aldehyde dehydrogenase superfamily. In addition, we established a NylD(1)/NylE(1) coupled system to quantify the aminotransferase activity and to enable the conversion of 6-aminohexaoate to adipate via adipate semialdehyde with a yield of > 90%. In the present study, we demonstrate that 6-aminohexanoate produced from polymeric nylon-6 and nylon oligomers (i.e., a mixture of 6-aminohexaoate oligomers) by nylon hydrolase (NylC) and 6-aminohexanoate dimer hydrolase (NylB) reactions are sequentially converted to adipate by metabolic engineering technology.

Due to the strict enantioselectivity of firefly luciferase, only D-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous L-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, L-luciferin is the biosynthetic precursor of D-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: L-enantiose-lective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of D-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient D-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for L-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.

We report here the 4.6-Mb genome sequence of a nylon oligomer-degrading bacterium, Arthrobacter sp. strain KI72. The draft genome sequence of strain KI72 consists of 4,568,574 bp, with a G+C content of 63.47%, 4,372 coding sequences (CDSs), 54 tRNAs, and six rRNAs.

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.DatabasePDB code : Hyb-24DNY-S-187 PDB code : Hyb-24DNY-A(187) PDB code : Hyb-24DNY-G(187) PDB code : Hyb-24DN-A(112)/Ahx complex PDB code : Hyb-24DNY-A(112)/Ahx complex PDB code : Hyb-24DNY-S(187)A(112)/Ahx complex PDB code : Hyb-24DNY-A(187)A(112)/Ahx complex PDB code : Hyb-24DNY-G(187)A(112)/Ahx complex

To elucidate how the nylon oligomer hydrolase (NylB) acquires its peculiar degradation activity towards non-biological amide bonds, we inspected the underlying enzymatic processes going from the induced-fit upon substrate binding to acylation. Specifically we investigated the mutational effects of two mutants, Y170F and D181G, indicated in former experiments as crucial systems because of their specific amino acid residues. Therefore, by adopting first-principles molecular dynamics complemented with metadynamics we provide a detailed insight into the underlying acylation mechanism. Our results show that while in the wild type (WT) the Tyr170 residue points the NH group towards the proton-acceptor site of an artificial amide bond, hence ready to react, in the Y170F this does not occur. The reason is ascribed to the absence of Tyr170 in the mutant, which is replaced by phenylalanine, which is unable to form hydrogen bond with the amide bond; thus, resulting in an increase in the activation barrier of more than 10 kcal mol(-1). Nonetheless, despite the lack of hydrogen bonding between the Y170F and the substrate, the highest free energy barrier for the induced-fit is similar to that of WT. This seems to suggest that in the induced-fit process, kinetics is little affected by the mutation. On the basis of additional structural homology analyses on the enzymes of the same family, we suggest that natural selection is responsible for the development of the peculiar hydrolytic activity of Arthrobacter sp. KI72.

Nylon hydrolase degrades various aliphatic nylons, including nylon-6 and nylon-66. We synthesized a nylon-66 copolymer (M (w) = 22,900, M (n) = 7,400), in which a part of an adipoyl unit (32 % molar ratio) of nylon-66 was replaced with a succinyl unit by interfacial polymerization. To quantify the reaction rate of the enzymatic hydrolysis of nylons at the surface of solid polymers, we prepared a thin layer of nylons on the bottom surface of each well in a polystyrene-based micro-assay plate. The thickness of the nylon layer was monitored by imaging analysis of the photographic data. More than 99 % of the copolymer with thicknesses of 260 nm (approximately 600 layers of polymer strands) were converted to water-soluble oligomers by nylon hydrolase (3 mg enzyme ml(-1)) at 30 A degrees C within 60 h. These results were further confirmed by TLC analysis of the reaction products and by assay of liberated amino groups in the soluble fractions. The degradation rate of the thin-layered nylon-6 was similarly analyzed. We demonstrate that this assay enables a quantitative evaluation of the reaction rate of hydrolysis at the interface between the solid and aqueous phases and a quantitative comparison of the degradability for various polyamides.

The active site of 6-aminohexanoate-dimer hydrolase, a nylon-6 byproduct-degrading enzyme with a beta-lactamase fold, possesses a Ser112/Lys115/Tyr215 catalytic triad similar to the one of penicillin-recognizing family of serine-reactive hydrolases but includes a unique Tyr170 residue. By using a reactive quantum mechanics/molecular mechanics (QM/MM) approach, we work out its catalytic mechanism and related functional/structural specificities. At variance with other peptidases, we show that the involvement of Tyr170 in the enzyme-substrate interactions is responsible for a structural variation in the substrate-binding state. The acylation via a tetrahedral intermediate is the rate-limiting step, with a free-energy barrier of similar to 21 kcal/mol, driven by the catalytic triad Ser112, Lys115, and Tyr215, acting as a nucleophile, general base, and general acid, respectively. The functional interaction of Tyr170 with this triad leads to an efficient disruption of the tetrahedral intermediate, promoting a conformational change of the substrate favorable for proton donation from the general acid.

A simple reaction procedure for chemiluminescence of firefly luciferin (D-luc) using n-propylphosphonic anhydride (T3P) is reported. A luminescent photon is produced as a result of one-pot reaction, only requiring mixing with the substrate carboxylic acid and T3P in the presence of a mild organic base.

Some strains belonging to the genera Citrobacter and Enterobacter have been reported to produce chitin/chitosan-like bioflocculants (BFs) from acetate. In this study, to investigate the distribution of the BF-producing potential in the genus Citrobacter and to screen stably and highly BF-producing strains, we obtained 36 Citrobacter strains from different culture collection centers, which were distributed among seven species in the genus, and tested for the flocculating activities of their culture supernatants using a kaolin suspension method. As a result, 21 strains belonging to C. freundii (17 strains in 23 strains tested), C. braakii (two in two), C. youngae (one in one), and C. werkmanii (one in two) showed flocculating activity, but this ability was limited to cells grown on acetate. Gas chromatography/mass spectrometry (GC/MS) analysis of the hydrolysates from the BFs of five selected strains indicated that they consisted of glucosamine and/or N-acetylglucosamine, such as the chitin/chitosan-like BF (BF04) produced by Citrobacter sp. TKF04 (Fujita et al. J Biosci Bioeng 89: 40-46, 2000). Gel filtration chromatography using a high-performance liquid chromatography system revealed that the molecular weight ranges of these BFs varied, but the average sizes were all above 1.66 x 10(6) Da.

We identified the critical amino acid residues for substrate recognition using two firefly luciferases from Pylocoeria miyako (PmL) and Hotaria parvura (HpL), as these two luciferase enzymes exhibit different activities toward ketoprofen. Specifically, PmL can catalyze the apparent enantioselective thioesterification reaction, while HpL cannot. By comparing the amino acid sequences around the active site, we identified two residues (I350 and M397 in PmL and F351 and S398 in HpL) that were different between the two enzymes, and the replacement of these amino acids resulted in changing the ketoprofen recognition pattern. The inactive HpL was converted to the active enzyme toward ketoprofen and vice versa for PmL. These residues also affected the enantioselectivity toward ketoprofen; however, the bioluminescent color was not affected. In addition, using molecular dynamics calculations, the replacement of these two amino acids induced changes in the state of hydrogen bonding between ketoprofen and the S349 side chain through the active site water. As S349 is not considered to influence color tuning, these changes specifically caused the differences in ketoprofen recognition in the enzyme.

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 angstrom. 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 angstrom. A rotation and translation search gave one clear solution containing two molecules per asymmetric unit.

Sphingomonas sp. NP5 can degrade a wide range of nonylphenol (NP) isomers that have widely contaminated aquatic environments as major endocrine-disrupting chemicals. To understand the biochemical and genetic backgrounds of NP degradation, a gene library of strain NP5 was constructed using a broad-host-range vector pBBR1MCS-2 and introduced into Sphingobium japonicum UT26. Several transformants accumulated reddish brown metabolites on agar plates dispersed with a mixture of NP isomers. Two different DNA fragments (7.6 and 9.3 kb) involved in the phenotype were isolated from the transformants. Sequence analysis revealed that both fragments contained an identical 1593 bp monooxygenase gene (nmoA), the predicted protein sequence of which showed 83% identity to the octylphenol-4-monooxygenase of Sphingomonas sp. PWE1. The nmoA gene in the 7.6 kb fragment was surrounded by an 1S21-type insertion sequence (IS) and 1S6100, while another in the 9.3 kb fragment was adjacent to an 1S66-type IS, suggesting that they have been acquired through multiple transposition events. A fast-growing recombinant Pseudomonas putida strain harbouring nmoA was constructed and used for degradation of a chemically synthesized NP isomer, 4-(1-ethyl-1-methylhexyl)phenol. This strain converted the isomer into hydroquinone stoichiometrically. 3-Methyl-3-octanol, probably originating from the alkyl side chain, was also detected as the metabolite. These results indicate that these two nmoA genes are involved in the NP degradation ability of strain NP5.

We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 angstrom-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 alpha beta beta alpha 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 T-m = 52 degrees C, enhanced the protein thermostability by 36 degrees C (T-m = 88 degrees C), whereas a single mutation (G111S or L137A) decreased the stability by similar to 10 degrees 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 degrees 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.

Measurement of thioesterification activities for dodecanoic acid (C12) and ketoprofen was done using five firefly luciferases, from Pyrocoelia miyako (PmL), Photinus pyralis (PpL), Luciola cruciata (LcL), Hotaria parvura (HpL), and Luciola mingrelica (LmL). Among these, PmL, PpL, and LcL showed the expected thioesterification activities toward both substrates. All the enzymes exhibited (R)-enantioselectivity toward ketoprofen, which had same tendency as firefly luciferase from Luciola lateralis (LUC-H). HpL and LmL, however, did not accept ketoprofen, although they had thioesterification activity toward C12. These results indicate that the substrate acceptance of luciferases for the thioesterification reaction varies dramatically relying on the origin of firefly. Hence we focused primarily on PmL and investigated the effect of pH on enzymatic activity. In addition, by determining the kinetic parameters at various pH values, we verified that the kat parameter contributed to the preferential enantioselectivity of this enzyme.

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 MNaCl. Diffraction data were collected from native and K2PtCl4-derivative crystals to resolutions of 2.00 and 2.20 angstrom, 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 angstrom. The anomalous difference Patterson map of the K2PtCl4-derivative crystal suggested that the space group was I222 rather than I2(1)2(1)2(1).

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. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.

We succeeded in the purification and gene cloning of a new enzyme, a-methyl carboxylic acid deracemizing enzyme 1 (MCAD1) from Brevibacterium ketoglutamicum KU1073, which catalyzes the (S)-enantioselective thioesterification reaction of 2-(4-chlorophenoxy)propanoic acid (CPPA). The cloned gene of MCAD1 contained an ORF of 1,623 bp, encoding a polypeptide of 540 amino acids. In combination with cofactors ATP, coenzyme A (CoASH), and Mg2+, MCAD1 demonstrated perfect enantioselectivity toward CPPA. The optimal. pH and temperature for reaction were found to be 7.25 and 30 degrees C. Under these conditions, the K-m and k(cat) values for (S)-CPPA were 0.92 +/- 0.17 mM and 0.28 +/- 0.026 s(-1) respectively. The results for substrate specificity revealed that MCAD1 had highest activity toward fatty acid tails with a medium chain-length (C-8). This result indicates that MCAD1 should be classified into a family of medium-chain acyl-CoA synthetase. This novel activity has never been reported for this family.

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 alpha/beta 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 angstrom resolution, suggested the following enzyme/substrate interactions: a Ser(174)-cis-Ser(150)-Lys(72) triad constitutes the catalytic center; the backbone N in Ala(171) and Ala(172) are involved in oxyanion stabilization; Cys(316)-S-gamma forms a hydrogen bond with nitrogen (Acd-N-7) 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 Ser(174)-cis-Ser(150)-Lys(72) is essential for catalysis. In contrast, substitutions at position 316 specifically affected Acd hydrolytic activity, suggesting that Cys(316) 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.

Promiscuous 6-aminohexanoate- linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a beta-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 (K-m = 15 mM) and turnover (k(cat) = 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-NH3+, and interaction between Ald-NH3+ and Gln27-O-epsilon 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-NH3+ (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 beta-lactamase fold.

A carboxylesterase with a beta-lactamase fold from Arthrobacter possesses a low level of hydrolytic activity (0.023 mu mol.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 (K(m) = 2.0 mm) than the wild-type Ald hydrolase from Arthrobacter (K(m) = 21 mm). In addition, the k(cat)/K(m) 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.

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/XyIS 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.

Microfluid filters were fabricated, which possessed 2,100 cylindrical through-bores (phi 40 mu m) in 200 mu m-thickness polymethylmethacrylate (PMMA) sheets (phi 3 mm), by deep X-ray lithography using synchrotron radiation. To evaluate the microfluid filters as a device for an immunoassay, we bound the goat anti-mouse immunogloblin G (IgG) antibody to the surface of the filters, and set the filters between reaction vessels stacked vertically in a microreactor. An enzyme-linked immunosorbent assay (ELISA) of mouse IgG using the goat anti-mouse IgG/horseradish-peroxidase (HRP) conjugate indicated that mouse IgG could be quantitatively detected in the range of 0-100 ng/ml, demonstrating the applicability of vertical microfluidic operation to the immunoassay.

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.

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 (Ny1C) 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.

Alkylcatechol 2,3-dioxygenase was purified from the cell extract of recombinant Escherichia coli JM109 harboring the alkylcatechol 2,3-dioxygenase gene (bupB) cloned from the butylphenol-degrading bacterium Pseudomonas putida MT4. The purified enzyme (BupB) showed relative metacleavage activities for the following catechols: catechol (100%), 4-methylcatechol (572%), 4-n-butylcatechol (185%), 4-n-hexylcatechol (53%), 4-n-heptylcatechol (45%), 4-n-nonylcatechol (10%), 4-tert-butylcatechol (0%), and 3-methylcatechol (33%). The kinetic parameters, namely, K(m) and V(max), for catechol, 4-methylcatechol, and 4-n-butylcatechol, were 23.4, 8.4, and 6.5 mu M and 25.8, 76.9, and 18.0 U mg(-1), respectively. These results suggest that BupB has broad substrate specificity for 4-n-alkylcatechols.

We developed an enzyme-linked immunosorbent assay for an endocrine disrupter, nonylphenol, using a microreactor composed of two reaction vessels stacked vertically through a microfluid filter. The filters constructed by deep X-ray lithography possessed 2100 through-bores (phi 40 x 200 mu m) in polymethylmethacrylate sheets (phi 3 mm), which are appropriate for biochemical reactions. Through the optimization of the immunoassay, nonylphenol was quantitatively detected at the range of 0.1-10 ng/ml.

We introduce a new application of firefly luciferase (EC 1.13.12.7). The firefly luciferases belong to a large superfamily that includes rat liver long-chain acyl-CoA syntbetase (LACS1). LACS1 is the enzyme that is involved in the deracernization process of 2-arylpropanoic acid and catalyzes the enantioselective thioester formation of R-acids. Based on the similarity of the reaction mechanisms and the sequences between firefly luciferase and LACS1, we predicted that firefly luciferase also has thioesterification activity toward 2-arylpropanoic acid. From an investigation using three kinds of luciferases from North American firefly and Japanese fireflies, we have confirmed that these luciferases exhibit an enantioselective thioester formation activity and the R-form is transformed to a thioester in preference to the S-form in the presence of ATP, Mg2+, and CoASH. The enantiomeric excesses of unreacted recovered acid and thioester were determined by chiral phase HPLC analysis and the resulting 2-arylpropanoyl-CoAs were identified by high resolution mass spectroscopy. The K-m, and k(cat) values of thermostable luciferase from Luciola lateralis (LUGH) toward ketoprofen were determined as 0.22 mM and 0.11 s(-1), respectively. The affinity of ketoprofen was almost the same of D-luciferin. In addition, the calculated E-value toward ketoprofen was approximately 20. These results suggest that LUGH could catalyze the kinetic resolution of 2-arylpropanoic acid efficiently and would be a new option for the preparation of optically active 2-substituted carboxylic acids.

Catechol 2,3-dioxygenase (C23O), a key enzyme in the meta-cleavage pathway of catechol metabolism, was purified from cell extract of recombinant Escherichia coli JM109 harboring the C23O gene (atdB) cloned from an aniline-degrading bacterium Acinetobacter sp. YAA. SDS-polyacrylamide gel electrophoresis and gel filtration chromatography analysis suggested that the enzyme (AtdB) has a molecular mass of 35 kDa as a monomer and forms a tetrameric structure. It showed relative meta-cleavage activities for the following catechols tested: catechol (100%), 3-methylcatechol (19%), 4-meth-ylcatechol (57%), 4-chlorocatechol: (46%), and 2,3-dihydroxybiphenyl (5%). To elevate the activity, a DNA selfshuffling experiment was carried out using the atdB gene. One mutant enzyme, named AtdBE286K, was obtained. It had one amino acid substitution, E286K, and showed 2.4-fold higher C23O activity than the wild-type enzyme at 100 mu M. Kinetic analysis of these enzymes revealed that the wild-type enzyme suffered from substrate inhibition at >2 mu M, while the mutant enzyme loosened substrate inhibition.

We performed X-ray crystallographic analyses of 6-aminohexanoate-dimer hydrolase (Hyb-24DN), an enzyme responsible for the degradation of nylon-6, an industry by-product, and of a complex between Hyb-24DNA- 12 (S112A-mutant of Hyb-24DN) and 6-aminohexanoate-linear dimer (Ald) at 1.58 angstrom and 1.4 angstrom resolution, respectively. In Hyb-24DN, Asp181-O-delta forms hydrogen bonds with Tyr170-O-eta, -two of the catalytic and binding amino acids, and a loop between Asn167 and Val177. This state is the so-called open form, allowing its substrate to bind in the space between the loop and catalytic residues. Upon substrate binding (in Hyb-24DN-A(112)/Ald complex), the loop is shifted 4.3 angstrom at Tyr170-C-alpha, and the side-chain of Tyr170 is rotated. By the combined effect, Tyr170-O-eta, moves a total of 10.5 angstrom, resulting in the formation of hydrogen bonds with the nitrogen of amide linkage in Ald (closed form). In addition, electrostatic interaction between Asp181-O-delta and the amino group in Ald stabilizes the substrate binding. We propose here that the enzyme catalysis proceeds according to the following steps: (i) Ald-induced transition from open to closed form, (ii) nucleophilic attack of Ser112 to Ald and formation of a tetrahedral intermediate, (iii) formation of acyl enzyme and transition to open form, (iv) deacylation. Amino acid substitutions reducing the enzyme/Ald interaction at positions 181 or 170 drastically decreased the Ald-hydrolytic activity, but had very little effect on esterolytic activity, suggesting that esterolytic reaction proceeds regardless of conversion. Present models illustrate why new activity against the nylon oligomer has evolved in an esterase with beta-lactamase folds, while retaining the original esterolytic functions. (C) 2007 Elsevier Ltd. All rights reserved.

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 angstrom. Diffraction data were collected from native and mercury(II) dichloride-derivative crystals to resolutions of 1.90 and 2.06 angstrom, respectively.

Alkylphenols (APs) are ubiquitous contaminants in aquatic environments and have endocrine disrupting and toxic effects on aquatic organisms. To investigate biodegradation mechanisms of APs, an AP degradation gene cluster was cloned from a butylphenol (BP)-degrading bacterium, Pseudomonas putida MT4. The gene cluster consisted of 13 genes named bupBA1A2A3A4A5A6CEH1FG From the nucleotide sequences, bupA1A2A3A4A5A6 were predicted to encode a multicomponent phenol hydroxylase (PH), whereas bupBCEHIFG were expected to encode meta-cleavage pathway enzymes. A partial sequence of a putative NtrC-type regulatory gene, bupR, was also found upstream of the gene bupB. This result indicates that APs can be initially oxidized into alkylcatechols (ACs), followed by the meta-cleavage of the aromatic rings. To confirm this pathway, AP degradation tests were carried out using the recombinant P putida KT2440 harboring the PH genes (bupA1A2A3A4A5A6). The recombinant strain oxidized 4-n-APs with an alkyl chain of up to C7 (<= C7) efficiently and also several BPs including those with an alkyl chain with some degree of branching. Therefore, it was found that PH had a broad substrate specificity for APs with a medium-length alkyl chain (C3-C7). Moreover, the cell extract of a recombinant Escherichia coli harboring bupB (a catechol 2,3-dioxygenase gene) converted 4-n-ACs with an alkyl chain of <= C9 into yellow meta-cleavage products with a maximum absorbance at 379 nm, indicating that the second step enzyme in this pathway is also responsible for the degradation of ACs with a medium-length alkyl chain. These results suggest that MT4 is a very useful strain in the biodegradation of a wide range of APs with a medium-length alkyl chain, which known nonylphenol-degrading Sphingomonas strains have never degraded.

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.

6-Aminohexanoate- dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII') that has only similar to 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 angstrom 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.