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The time profiles of active cell ratios depended on the growth phase and the absence of some lytic transglycosylases of E. coli. Significant cell damage was not found on the autolysis inhibition condition.

Cyclohexanecarboxylate (CHCA) is formed by oxidative microbial degradation of n-alkylcycloparaffins and anaerobic degradation of benzoate, and also known to be a synthetic intermediate or the starter unit of biosynthesis of cellular constituents and secondary metabolites. Although two degradation pathways have been proposed, genetic information has been limited to the β-oxidation-like pathway. In this study, we identified a gene cluster, designated chcC1XTC2B1B2RAaAbAc, that is responsible for the CHCA aromatization pathway in Sinomonas (formerly Corynebacterium) cyclohexanicum strain ATCC 51369. Reverse transcription-PCR analysis indicated that the chc gene cluster is inducible by CHCA and that it consists of two transcriptional units, chcC1XTC2B1B2R and chcAaAbAc. Overexpression of the various genes in Escherichia coli, and purification of the recombinant proteins led to the functional characterization of ChcAaAbAc as subunits of a cytochrome P450 system responsible for CHCA hydroxylation; ChcB1 and ChcB2 as trans-4-hydroxyCHCA and cis-4-hydroxyCHCA dehydrogenases, respectively; ChcC1 was identified as a 4-oxoCHCA desaturase containing a covalently bound FAD; and ChcC2 was identified as a 4-oxocyclohexenecarboxylate desaturase. The binding constant of ChcAa for CHCA was found to be 0.37 mM. Kinetic parameters established for ChcB1 indicated that it has a high catalytic efficiency towards 4-oxoCHCA compared to trans- or cis-4-hydroxyCHCA. The Km and Kcat values of ChcC1 for 4-oxoCHCA were 0.39 mM and 44 s−1, respectively. Taken together with previous work on the identification of a pobA gene encoding a 4-hydroxybenzoate hydroxylase, we have now localized the remaining set of genes for the final degradation of protocatechuate before entry into the tricarboxylic acid cycle.

Paraburkholderia terrae strain KU-15 has been investigated for its ability to degrade 2-nitrobenzoate. Here, we report the complete 10,422,345-bp genome of this microorganism, which consists of six circular replicons containing 9,483 protein-coding sequences. The genome carries genes that are potentially responsible for 2-nitrobenzoate and 4-nitirobenzoate degradation.

The fungus Exophiala jeanselmei strain KUFI-6N produces a unique cycloalkanone monooxygenase (ExCAMO) that displays an uncommon substrate spectrum of Baeyer–Villiger oxidation of 4-10-membered ring ketones. In this study, we aimed to identify and sequence the gene encoding ExCAMO from KUFI-6N and overexpress the gene in Escherichia coli. We found that the primary structure of ExCAMO is most closely related to the cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011, with 54.2% amino acid identity. ExCAMO was functionally expressed in E. coli and its substrate spectrum and kinetic parameters were investigated. Substrate profiling indicated that ExCAMO is unusual among known Baeyer–Villiger monooxygenases owing to its ability to accept a variety of substrates, including C4-C12 membered ring ketones. ExCAMO has high affinity and catalytic efficiency toward cycloalkanones, the highest being toward cyclohexanone. Five other genes encoding Baeyer–Villiger monooxygenases were also cloned and expressed in E. coli.

The degradation pathways in microorganisms for piperidine, a secondary amine with various applications, are not yet fully understood, especially in non-Mycobacterium species. In this study, we have identified a piperidine-degrading isolate (KU43P) from a soil sample collected in a cultivation field in Osaka, Japan, and characterized its mechanisms of piperidine degradation, thereby furthering current understanding of the process. The genome of isolate KU43P consists of a 5,869,691-bp circular chromosome with 62.67% GC content and with 5,294 predicted protein-coding genes, 77 tRNA genes, and 22 rRNA genes. 16S rRNA gene sequence analysis and average nucleotide identity analysis suggest that the isolate is a novel species of the Pseudomonas putida group in the genus Pseudomonas. The genomic region encoding the piperidine degradation pathway, designated as the pip gene cluster, was identified using transposon mutagenesis and reverse transcription polymerase chain reaction. Deletion analyses of pipA, which encodes a glutamine synthetase (GS)-like protein, and pipBa, which encodes a cytochrome P450 monooxygenase, indicate that pipA and pipBa are involved in piperidine metabolism and suggest that pipA is involved in the first step of the piperidine metabolic pathway. Escherichia coli whole cells overexpressing PipA converted piperidine and glutamate to γ-glutamylpiperidide, and crude cell extract enzyme assays of PipA showed that this reaction requires ATP and Mg2+. These results clearly show that pipA encodes γ-glutamylpiperidide synthetase and that piperidine is first glutamylated and then hydroxylated in the piperidine degradation pathway of Pseudomonas sp. strain KU43P. This study has filled a void in the general knowledge of the microbial degradation of amine compounds.

Nanostructures exhibit a bactericidal effect owing to physical interaction with the bacterial cell envelope. Here, we aimed to identify the mechanism underlying the bactericidal effect of nanostructures based on bacterial autolysis, in contrast to previous reports focusing on structural characteristics. The time profiles of active cell ratios of the

Cytochrome P450 monooxygenases play important roles in metabolism. Here, we report the identification and biochemical characterization of P450CHC, a novel self-sufficient cytochrome P450, from cyclohexanecarboxylate-degrading Paraburkholderia terrae KU-64. P450CHC was found to comprise a [2Fe-2S] ferredoxin domain, NAD(P)H-dependent FAD-containing reductase domain, FCD domain, and cytochrome P450 domain (in that order from the N terminus). Reverse transcription–polymerase chain reaction results indicated that the P450CHC-encoding chcA gene was inducible by cyclohexanecarboxylate. chcA overexpression in Escherichia coli and recombinant protein purification enabled functional characterization of P450CHC as a catalytically self-sufficient cytochrome P450 that hydroxylates cyclohexanecarboxylate. Kinetic analysis indicated that P450CHC largely preferred NADH (Km = 0.011 m m) over NADPH (Km = 0.21 m m). The Kd, Km, and kcat values for cyclohexanecarboxylate were 0.083 m m, 0.084 m m, and 15.9 s−1, respectively. The genetic and biochemical analyses indicated that the physiological role of P450CHC is initial hydroxylation in the cyclohexanecarboxylate degradation pathway.

The degradation pathways in microorganisms for piperidine, a secondary amine with various applications, are not yet fully understood, especially in non-Mycobacterium species. In this study, we have identified a piperidine-degrading isolate (KU43P) from a soil sample collected in a cultivation field in Osaka, Japan, and characterized its mechanisms of piperidine degradation, thereby furthering current understanding of the process. The genome of isolate KU43P consists of a 5,869,691-bp circular chromosome with 62.67% GC content and with 5,294 predicted protein-coding genes, 77 tRNA genes, and 22 rRNA genes. 16S rRNA gene sequence analysis and average nucleotide identity analysis suggest that the isolate is a novel species of the Pseudomonas putida group in the genus Pseudomonas. The genomic region encoding the piperidine degradation pathway, designated as the pip gene cluster, was identified using transposon mutagenesis and reverse transcription polymerase chain reaction. Deletion analyses of pipA, which encodes a glutamine synthetase (GS)-like protein, and pipBa, which encodes a cytochrome P450 monooxygenase, indicate that pipA and pipBa are involved in piperidine metabolism and suggest that pipA is involved in the first step of the piperidine metabolic pathway. Escherichia coli whole cells overexpressing PipA converted piperidine and glutamate to γ-glutamylpiperidide, and crude cell extract enzyme assays of PipA showed that this reaction requires ATP and Mg

Here, we report the complete genome sequence of Mameliella alba strain KU6B, a bacterium newly isolated from seawater of Boso Peninsula in Japan that is capable of utilizing cyclohexylamine. The complete genome contained a 5,386,988-bp circular chromosome and three circular plasmids of 256,516, 112,434, and 76,727 bp.

Pseudomonas sp. (formerly Pseudomonas fluorescens) strain KUIN-1 is an ice-nucleating bacterium that was isolated from the leaves of field beans (Phaseolus vulgaris L.). This microorganism can release cell-free ice nucleation proteins and shows cold shock-induced freezing tolerance. Here, we report the 6,028,589-bp complete genome sequence of Pseudomonas sp. KUIN-1.

Besides an industrial pollutant, 2,4-dinitrophenol (DNP) has been used illegally as a weight loss drug that had claimed human lives. Little is known about the metabolism of DNP, particularly among Gram-negative bacteria. In this study, two non-contiguous genetic loci ofParaburkholderia(formerlyBurkholderia) sp. strain KU-46 genome were identified and four key initial genes (dnpA,dnpB, anddnpC1C2) were characterized to provide molecular and biochemical evidence for the degradation of DNP via the formation of 4-nitrophenol (NP), a pathway that is unique among DNP utilizing bacteria. Reverse transcription PCR analysis indicated that thednpAgene encoding the initial hydride transferase (28 kDa), and thednpBgene encoding a nitrite-eliminating enzyme (33 kDa), are inducible by DNP and the two genes are organized in an operon. Purified DnpA and DnpB from overexpression clones inEscherichia colieffected the transformation of DNP to NP via the formation of hydride-Meisenheimer complex of DNP. The function of DnpB appears new since all homologs of DnpB sequences in the protein database are annotated as putative nitrate ABC transporter substrate-binding proteins. The gene cluster responsible for the degradation of DNP after NP formation was designateddnpC1C2DXFER.DnpC1 and DnpC2 were functionally characterized as the respective FAD reductase and oxygenase components of the two-component NP monooxygenase. Both NP and 4-nitrocatechol were shown to be substrates, producing hydroquinone and hydroxyquinol, respectively. Elucidation of thehqdA1A2BCDgene cluster allows the delineation of the final degradation pathway of hydroquinone to ß-ketoadipate prior to its entry to the tricarboxylic acid cycle.ImportanceThis study fills a gap in our knowledge and understanding of the genetic basis and biochemical pathway for the degradation of 2,4-dinitrophenol (DNP) in Gram-negative bacteria, represented by the prototypicalParaburkholderiasp. strain KU-46 that metabolizes DNP through the formation of 4-nitrophenol, a pathway unseen by other DNP utilizers. The newly cloned genes could serve as DNA probes in biomonitoring as well as finding application in new biocatalyst development to access green chemicals. By and large, knowledge of the diverse strategies used by microorganisms to degrade DNP will contribute to the development of bioremediation solutions since DNP is an industrial pollutant used widely in the chemical industry for the synthesis of pesticides, insecticides, sulfur dyes, wood preservatives, and explosives, etc. (119 words)

Seven xylene-utilizing bacterial strains were isolated from seawater collected off the coast of Japan. Analysis of 16S rRNA gene sequences indicated that six isolates were most closely related to the marine bacterial genera Alteromonas, Marinobacter or Aestuariibacter. The sequence of the remaining strain, KU68FT, showed low similarity to the 16S rRNA gene sequences of known bacteria with validly published names, the most similar species being Maricurvus nonylphenolicus strain KU41ET (92.6% identity). On the basis of physiological, chemotaxonomic and phylogenetic data, strain KU68FT is suggested to represent a novel species of a new genus in the family Cellvibrionaceae of the order Cellvibrionales within the Gammaproteobacteria, for which the name Halioxenophilus aromaticivorans gen. nov., sp. nov. is proposed. The type strain of Halioxenophilus aromaticivorans is KU68FT (=JCM 19134T = KCTC 32387T). PCR and sequence analysis revealed that strain KU68FT possesses an entire set of genes encoding the enzymes for the upper xylene methyl-monooxygenase pathway, xylCMABN, resembling the gene set of the terrestrial Pseudomonas putida strain mt-2., This work was financially supported in part by the Kansai University Fund for Supporting Young Scholars (2016).

Human α-amino-β-carboxymuconate-e-semialdehyde decarboxylase determines the fate of tryptophan metabolites in the kynurenine pathway by controlling the quinolinate levels for de novo nicotinamide adenine dinucleotide biosynthesis. The unstable nature of its substrate has made gaining insight into its reaction mechanism difficult. Our electron paramagnetic resonance (EPR) spectroscopic study on the Cu-substituted human enzyme suggests that the native substrate does not directly ligate to the metal ion. Substrate binding did not result in a change of either the hyperfine structure or the super-hyperfine structure of the EPR spectrum. We also determined the crystal structure of the human enzyme in its native catalytically active state (at 1.99 A resolution), a substrate analogue-bound form (2.50 A resolution), and a selected active site mutant form with one of the putative substrate binding residues altered (2.32 A resolution). These structures illustrate that each asymmetric unit contains three pairs of dimers. Consistent with the EPR findings, the ligand-bound complex structure shows that the substrate analogue does not directly coordinate to the metal ion but is bound to the active site by two arginine residues through noncovalent interactions. Proteins 2015; 83:178–187. © 2014 Wiley Periodicals, Inc.

A cyclohexylamine oxidase (CHAO) of bacterial origin was previously shown to be a potentially useful catalyst in the deracemization of racemic primary amines. To further explore the properties and application of this enzyme, five single-amino acid substitution mutants (L199A, M226A, Y321A, Y321F, and L353M) were created based on superimposition of the tertiary structure of CHAO and the monoamine oxidase (MAO) B homolog. The substrate specificity of the purified wild-type and five mutant enzymes were examined towards 38 structurally diverse amines. All the enzymes exhibited better activity for primary amines than secondary and tertiary amines and in general exhibited high stereoselectivity. Among the mutant enzymes, M226A displayed an enhanced activity (5-400 %) towards most substrates, and L353M showed 7-445 % higher activity towards primary aliphatic amines with cycloalkane or aromatic moieties. Kinetic parameters revealed that both Y321 mutants showed higher catalytic efficiency towards cyclooctanamine, whereas the wild-type CHAO (wt CHAO) was most efficient towards cyclohexylamine. The wt CHAO or variant L353M in combination with a borane-ammonia complex as reducing agent was applied to the deracemization of 1-aminotetraline to give the (R)-enantiomer, a precursor of an antidepressant drug Norsertraline, in good yield (73-76 %), demonstrating their application potential in chiral amine synthesis.

The previously reported crystal structures of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) show a five-coordinate Zn(II)(His)(3)(Asp)(OH(2)) active site. The water ligand is H-bonded to a conserved His228 residue adjacent to the metal center in ACMSD from Pseudomonas fluorescens (PfACMSD). Site-directed mutagenesis of His228 to tyrosine and glycine in this study results in a complete or significant loss of activity. Metal analysis shows that H228Y and H228G contain iron rather than zinc, indicating that this residue plays a role in the metal selectivity of the protein. As-isolated H228Y displays a blue color, which is not seen in wild-type ACMSD. Quinone staining and resonance Raman analyses indicate that the blue color originates from Fe(III)-tyrosinate ligand-to-metal charge transfer. Co(II)-substituted H228Y ACMSD is brown in color and exhibits an electron paramagnetic resonance spectrum showing a high-spin Co(II) center with a well-resolved (59)Co (I = 7/2) eight-line hyperfine splitting pattern. The X-ray crystal structures of as-isolated Fe-H228Y (2.8 Å) and Co-substituted (2.4 Å) and Zn-substituted H228Y (2.0 Å resolution) support the spectroscopic assignment of metal ligation of the Tyr228 residue. The crystal structure of Zn-H228G (2.6 Å) was also determined. These four structures show that the water ligand present in WT Zn-ACMSD is either missing (Fe-H228Y, Co-H228Y, and Zn-H228G) or disrupted (Zn-H228Y) in response to the His228 mutation. Together, these results highlight the importance of His228 for PfACMSD's metal specificity as well as maintaining a water molecule as a ligand of the metal center. His228 is thus proposed to play a role in activating the metal-bound water ligand for subsequent nucleophilic attack on the substrate.

A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ 3 -4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ 3 -4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP + at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP + . A comparison of several crystal forms of OTEMO bound to FAD and NADP + revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ 3 -4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency ( k cat / K m ) favors 2- n -hexyl cyclopentanone (4.3 × 10 5 M −1 s −1 ) as a substrate, although its affinity ( K m = 32 μM) was lower than that of the CoA-activated substrate ( K m = 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

An aerobic, Gram-negative bacterial strain, designated KU27E1(T), which degrades phthalate and dimethylphthalate, was isolated from seawater obtained from the coastal region of Ishigaki Island, Japan. Cells are motile rods with polar flagella. Strain KU27E1(T) grew at 15-30°C, pH 6.0-8.0, in the presence of 1.0-2.0% (w/v) NaCl. The 16S rRNA gene sequence analysis revealed that this strain was affiliated with the family Rhodobacteraceae in the class Alphaproteobacteria, and was most closely related to Tropicibacter naphthalenivorans (96.8%). The predominant respiratory lipoquinone was ubiquinone-10, and the major cellular fatty acid was C(18:1)ω7c (88.5%). The G+C content of genomic DNA was 58.7 mol%. Based on the physiological, chemotaxonomic, and phylogenetic data, strain KU27E1(T) is suggested to represent a novel species of the genus Tropicibacter, for which the name Tropicibacter phthalicus sp. nov. is proposed. The type strain of Tropicibacter phthalicus is designated as KU27E1(T) (=JCM 17793(T) = KCTC 23703(T)).

A novel aerobic, Gram-negative bacterial strain, designated KU41E(T), which degrades p-n-nonylphenol, was isolated from seawater obtained from the coastal region of Ishigaki Island, Japan. Cells are motile, curved rods with a single polar flagellum. Strain KU41E(T) grew at 20-35 °C, pH 7.0-8.0, in the presence of 1.0-4.0% NaCl. The predominant respiratory lipoquinone was ubiquinone-8, and the major cellular fatty acids were summed feature 3 (C(15:0) iso 2OH and/or C(16:1) ω7c, 28.4%), C(18:1) ω7c (19.8%), and C(16:0) (17.0%). The DNA G + C content was 48.6 mol%. The 16S rRNA gene sequence analysis indicated that strain KU41E(T) is affiliated with the order Alteromonadales within the class Gammaproteobacteria and is most closely related to Pseudoteredinibacter isoporae SW-11(T) (93.6% similarity) and Teredinibacter turnerae T7902(T) (91.9% similarity). On the basis of physiological, chemotaxonomic, and phylogenetic data, strain KU41E(T) is suggested to represent a novel species of a new genus, for which the name Maricurvus nonylphenolicus gen. nov., sp. nov. is proposed. The type strain of M. nonylphenolicus is KU41E(T) (=JCM 17778(T)).

Eleven phthalate-degrading bacterial strains were isolated from seawater collected off the coast of Japan. The isolates were found to be most closely related to the marine bacterial genera Alteromonas, Citreicella, Marinomonas, Marinovum, Pelagibaca, Rhodovulum, Sulfitobacter, Thalassobius, Thalassococcus, Thalassospira, and Tropicibacter. For the first time, members of these genera were shown to be capable of growth on phthalate. The plate assay for visual detection of phthalate dioxygenase activity and PCR detection of a possible gene encoding 4,5-dihydroxyphthalate decarboxylase indicated that phthalate is degraded via 4,5-dihydroxyphthalate to protocatechuate in all the isolates.

The first crystal structure of a type II Baeyer–Villiger monooxygenase reveals a different ring orientation of its FMN cofactor compared with other related bacterial luciferase-family enzymes., The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer–Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily.

Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O(2) as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP(+) in two distinct states, to resolutions of 2.3 and 2.2 A. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

A gram-negative bacterium, strain KU-46, was isolated from agricultural soil contaminated with pesticides and was found to utilize 2,4-dinitrophenol as the sole source of carbon and nitrogen. Based on 16S rRNA gene sequence analysis and its morphological, biochemical, and physiological characteristics, strain KU-46 was identified as a Burkholderia sp. Metabolite analyses by HPLC and liquid chromatography-MS indicated that 4-nitrophenol, 1,4-benzoquinone, and nitrite are the intermediates of 2,4-dinitrophenol metabolism, and 2,4-dinitrophenol is metabolized via 4-nitrophenol to 1,4-benzoquinone by strain KU-46. The 2,4-dinitrophenol degradation pathway enzymes are induced by both 2,4-dinitrophenol and 4-nitrophenol.

Bacterial strain KU-15, identified as a Burkholderia terrae by 16S rRNA gene sequence analysis, was one of 11 new isolates that grew on 2-nitrobenzoate as sole source of carbon and nitrogen. Strain KU-15 was also found to grow on anthranilate, 4-nitrobenzoate, and 4-aminobenzoate. Whole cells of strain KU-15 were found to accumulate ammonia in the medium, indicating that the degradation of 2-nitrobenzoate proceeds through a reductive route. Metabolite analyses by high-performance liquid chromatography indicated that 3-hydroxyanthranilate, anthranilate, and catechol are intermediates of 2-nitrobenzoate metabolism in strain KU-15. Enzyme studies suggested that 2-nitrobenzoate degradation occurs via the formation of 2-hydroxylaminobenzoate and that the pathway branches at this point to form two different aromatic intermediates: anthranilate and 3-hydroxyanthranilate. PCR amplifications and DNA sequencing revealed DNA fragments encoding a polypeptide homologous to 2-amino-3-carboxymuconate 6-semialdehyde decarboxylase and anthranilate 1,2-dioxygenase.

Aldehydes are ubiquitous intermediates in metabolic pathways, and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation which can decay into neuroactive compounds that have been associated with numerous neurological diseases, such as quinolinic acid (QUIN). An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, which is responsible for 'disarming' the final aldehydic intermediate, has yet to be studied at the molecular level. Its activity steers the vast majority of metabolic flux to further catabolism and prevents overproduction of a metabolic dead-end, picolinic acid. In this presentation we will show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms, two of which are covalently-bound, substrate-enzyme adducts. The crystal- and solution-state spectroscopic data along with the crystal structures also reveal that the substrate must undergo an isomer...

Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation., Aldehydes are common intermediates in enzymatic pathways, but their high reactivity can make them difficult to observe. Here, the authors study the mechanism of aldehyde deactivation in a dehydrogenase, showing a key E/Z isomerization and observing a thiohemiacetal intermediate by crystal structure analysis.

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer–Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid ofPseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in acisconformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily.

The enzymatic activity of Pseudomonas fluorescens alpha-amino-beta-carboxymuconic-epsilon-semialdehyde decarboxylase (ACMSD) is critically dependent on a transition metal ion [Li, T., Walker, A. L., Iwaki, H., Hasegawa, Y., and Liu, A. (2005) J. Am. Chem. Soc. 127, 12282-12290]. Sequence analysis in this study further suggests that ACMSD belongs to the amidohydrolase superfamily, whose structurally characterized members comprise a catalytically essential metal cofactor. To identify ACMSD's metal ligands and assess their functions in catalysis, a site-directed mutagenesis analysis was conducted. Alteration of His-9, His-177, and Asp-294 resulted in a dramatic loss of enzyme activity, substantial reduction of the metal-binding ability, and an altered metallocenter electronic structure. Thus, these residues are confirmed to be the endogenous metal ligands. His-11 is implicated in metal binding because of the strictly conserved HxH motif with His-9. Mutations at the 228 site yielded nearly inactive enzyme variants H228A and H228E. The two His-228 mutant proteins, however, exhibited full metal-binding ability and a metal center similar to that of the wild-type enzyme as shown by EPR spectroscopy. Kinetic analysis on the mutants indicates that His-228 is a critical catalytic residue along with the metal cofactor. Since the identified metal ligands and His-228 are present in all known ACMSD sequences, it is likely that ACMSD proteins from other organisms contain the same cofactor and share similar catalytic mechanisms. ACMSD is therefore the first characterized member in the amidohydrolase superfamily that represents a C-C breaking activity.

The enzyme alpha-amino-beta-carboxy-muconic-epsilon-semialdehyde decarboxylase (ACMSD) plays an important role in the biodegradation of 2-nitrobenzoic acid in microorganisms and in tryptophan catabolism in humans. We report that the overexpressed ACMSD enzyme from Pseudomonas fluorescens requires a divalent metal, such as Co(II), Fe(II), Cd(II), or Mn(II), for catalytic activity and that neither a redox reagent nor an organic cofactor is required for the catalytic function. The metal ions can be taken up in either cell or cell-free preparations for generating the active form of ACMSD. The kinetic parameters and enzyme specific activity are shown to depend on the metal ion present in the enzyme, suggesting a catalytic role of the metal center. EPR spectrum of Co(II)-ACMSD provides a high-spin (S = 3/2 mononuclear metal ion in a non-heme, noncorrinoid environment with a mixed nitrogen/oxygen ligand field. We observe hyperfine interactions due to 59Co nucleus at temperatures below 5 K but not at higher temperatures. Ten hyperfine lines are present in the g(perpendicular) region, and three equivalent nitrogen hyperfine couplings are required to simulate the resonances in the EPR spectrum. The results for the metal binding site are also assessed using the copper-substituted enzyme, and the EPR spectral assignments for both cobalt and copper proteins give strong support for a distorted trigonal bipyramidal geometry of the metal center. Ultimately, these results suggest for the first time that ACMSD is a metal-dependent enzyme that catalyzes a novel nonoxidative decarboxylation.

A DNA fragment that carries the gene encoding 4-hydroxybenzoate 3-hydroxylase (pobA) together with genes encoding a potential regulator (pobR) and a potential transporter (pobK) was cloned from "Corynebacterium cyclohexanicum" strain ATCC 51369, which is herein reclassified as belonging to the genus Arthrobacter. Nucleotide sequencing revealed that the deduced amino acid sequence encoded by the Arthrobacter pobA gene exhibits 42.0-46.7% identity with that of gram-negative bacteria. The gene organization of the pob cluster differs from that of gram-negative bacteria. The pobA gene product (PobA), expressed in Escherichia coli, preferred NADH over NADPH similar to 4-hydroxybenzoate 3-hydroxylase of ATCC 51369. The Arthrobacter pobA gene was inactivated by insertion of pK19mob. The resultant mutant strain, POBA1, grew on neither cyclohexanecarboxylate nor 4-hydroxybenzoate. These results strongly suggest that the cloned pobA gene plays an essential role in the catabolism not only of 4-hydroxybenzoate but also of cyclohexanecarboxylate in strain ATCC 51369.

Cyclopentanone 1,2-monooxygenase, a flavoprotein produced by Pseudomonas sp. strain NCIMB 9872 upon induction by cyclopentanol or cyclopentanone (M. Griffin and P. W. Trudgill, Biochem. J. 129:595-603, 1972), has been utilized as a biocatalyst in Baeyer-Villiger oxidations. To further explore this biocatalytic potential and to discover new genes, we have cloned and sequenced a 16-kb chromosomal locus of strain 9872 that is herein reclassified as belonging to the genus Comamonas . Sequence analysis revealed a cluster of genes and six potential open reading frames designated and grouped in at least four possible transcriptional units as ( orf11-orf10-orf9)-(cpnE-cpnD-orf6-cpnC)-(cpnR-cpnB-cpnA)-(orf3-orf4 [partial 3′ end] ) . The cpnABCDE genes encode enzymes for the five-step conversion of cyclopentanol to glutaric acid catalyzed by cyclopentanol dehydrogenase, cyclopentanone 1,2-monooxygenase, a ring-opening 5-valerolactone hydrolase, 5-hydroxyvalerate dehydrogenase, and 5-oxovalerate dehydrogenase, respectively. Inactivation of cpnB by using a lacZ -Km r cassette resulted in a strain that was not capable of growth on cyclopentanol or cyclopentanone as a sole carbon and energy source. The presence of σ 54 -dependent regulatory elements in front of the divergently transcribed cpnB and cpnC genes supports the notion that cpnR is a regulatory gene of the NtrC type. Knowledge of the nucleotide sequence of the cpn genes was used to construct isopropyl-β-thio- d -galactoside-inducible clones of Escherichia coli cells that overproduce the five enzymes of the cpn pathway. The substrate specificities of CpnA and CpnB were studied in particular to evaluate the potential of these enzymes and establish the latter recombinant strain as a bioreagent for Baeyer-Villiger oxidations. Although frequently nonenantioselective, cyclopentanone 1,2-monooxygenase was found to exhibit a broader substrate range than the related cyclohexanone 1,2-monooxygenase from Acinetobacter sp. strain NCIMB 9871. However, in a few cases opposite enantioselectivity was observed between the two biocatalysts.

A bacterial strain KU-7, identified as a Pseudomonas fluorescens by 16S rDNA sequencing, was one of the 12 new isolates that are able to grow on 2-nitrobenzoate as a sole source of carbon, nitrogen, and energy. Resting cells of KU-7 were found to accumulate ammonia in the medium indicating that degradation of 2-NBA proceeds through a reductive route. Metabolite analyses by thin layer chromatography and high pressure liquid chromatography indicated that 3-hydroxyanthranilate is an intermediate of 2-nitrobenzoate metabolism in KU-7 cells. This offers an alternative route to 2-nitrobenzoate metabolism since anthranilate (2-aminobenzoate) or catechol were detected as intermediates in other bacteria. Crude extracts of KU-7 cells converted 2-nitrobenzoate to 3-hydroxyanthranilate with oxidation of 2 mol of NADPH. Ring cleavage of 3-hydroxyanthranilate produced a transient yellow product, identified as 2-amino-3-carboxymuconic 6-semialdehyde, that has a maximum absorbance at 360 nm. The initial enzymes of the 2-nitrobenzoate degradation pathway were found to be inducible since succinate-grown cells produced very low enzyme activities. A pathway for 2-nitrobenzoate degradation in KU-7 was proposed.

Two novel aerobic p-n-nonylphenol-degrading bacterial strains were isolated from seawater obtained from the coastal region of Ogasawara Islands, Japan. The 16S rRNA gene sequence analysis indicated that the strains are affiliated with the order Alteromonadales within the class Gammaproteobacteria. One isolate, strain KU41G2, is most closely related to Maricurvus nonylphenolicus (99.2 % similarity), and is tentatively identified as M. nonylphenolicus. The other isolate, strain KU41G(T), is also most closely related to M. nonylphenolicus; however, the 16S rRNA gene sequence similarity was only 94.7 %. Cells of strain KU41G(T) are Gram-negative rods with a single polar flagellum. The predominant respiratory lipoquinone was ubiquinone-8, and the major cellular fatty acids were C17:1 ω8c (24.2 %); C15:0 iso 2-OH; and/or C16:1 ω7c (16.3 %), C15:0 (10.3 %), C11:0 3-OH (9.5 %), C9:0 3-OH (6.7 %), C10:0 3-OH (6.4 %), and C18:1 ω7c (5.5 %). The DNA G+C content was 53.3 mol%. On the basis of physiological, chemotaxonomic, and phylogenetic data, strain KU41G(T) is suggested to represent a novel species of a new genus, for which we propose the name Pseudomaricurvus alkylphenolicus gen. nov., sp. nov. The type strain of P. alkylphenolicus is KU41G(T) (=JCM 19135(T) = KCTC 32386(T)).

A novel aerobic, Gram-negative, non-motile, pleomorphic, and rod-shaped bacterium designated KU5D5(T) was isolated from seawater that was obtained from the coastal region of the Goto Islands, Japan, on the basis of its ability to utilize cyclohexylacetate as the sole source of carbon and energy. Strain KU5D5(T) grew at pH 6.0-8.0 and 10-35 °C in the presence of 1.0-5.0 % (w/v) NaCl. Analysis of the 16S rRNA gene sequence revealed that this strain was affiliated to the family Rhodobacteraceae in the class Alphaproteobacteria and was related most closely to Lutimaribacter saemankumensis (96.6 % similarity) and Oceanicola pacificus (96.6 %). The predominant respiratory lipoquinone was ubiquinone-10 and the major cellular fatty acids were C(18:1) ω7c (66.7 %), C(16:0) (7.7 %), C(12:1) 3-OH (6.1 %), and C(17:0) (6.1 %). The DNA G+C content was 58.9 mol %. On the basis of physiological, chemotaxonomic, and phylogenetic data, strain KU5D5(T) is suggested to represent a novel species of the genus Lutimaribacter, for which the name Lutimaribacter litoralis sp. nov. is proposed. It is also proposed that O. pacificus should be transferred to the genus Lutimaribacter as Lutimaribacter pacificus comb. nov. The type strain of L. litoralis is KU5D5(T) (=JCM 17792(T) = KCTC 23660(T)) and the type strain of L. pacificus is W11-2B(T) (=CCTCC AB 208224(T) = LMG 24619(T) = MCCC 1A01034(T)).

Trichosporon cutaneum strain KUY-6A, a cyclohexanecarboxylic acid-utilizing yeast, was able to grow on cyclopentanol (CPOL) as a sole source of carbon and energy. Growth experiments revealed the strain, KUY-6A, could utilize up to 42 mM of CPOL with an optimum at 24 mM. Optimal growth was found between pH 4.0 to 9.0. The generation time under optimal growth conditions on CPOL was 3.0 h. Analysis indicated that cyclopentanone (CPON) and glutaric acid were intermediates of CPOL metabolism in strain KUY-6A. The results of growth and enzyme experiments are consistent with the degradation of CPOL via CPON, 5-valerolactone, 5-hydroxyvaleric acid, and glutaric acid.

Cyclohexylamine oxidase (CHAO) is a flavoprotein first described in Brevibacterium oxydans strain IH-35A that carries out the initial step of the degradation of the industrial chemical cyclohexylamine to cyclohexanone. We have cloned and expressed in Escherichia coli the CHAO-encoding gene (chaA) from B. oxydans, purified CHAO and determined the structures of both the holoenzyme form of the enzyme and a product complex with cyclohexanone. CHAO is a 50 kDa monomer with a PHBH fold topology. It belongs to the flavin monooxygenase family of enzymes and exhibits high substrate specificity for alicyclic amines and sec-alkylamines. The overall structure is similar to that of other members of the flavin monooxygenase family, but lacks either of the C- or N-terminal extensions observed in these enzymes. Active site features of the flavin monooxygenase family are conserved in CHAO, including the characteristic aromatic cage. Differences in the orientations of residues of the CHAO aromatic cage result in a substrate-binding site that is more open than those of its structural relatives. Since CHAO has a buried hydrophobic active site with no obvious route for substrates and products, a random acceleration molecular dynamics simulation has been used to identify a potential egress route. The path identified includes an intermediate cavity and requires transient conformation changes in a shielding loop and a residue at the border of the substrate-binding cavity. These results provide a foundation for further studies with CHAO aimed at identifying features determining substrate specificity and for developing the biocatalytic potential of this enzyme. © 2013 Mirza et al.

Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp NCIMB 9871 expressed in baker's yeast and in E. coli and cyclopentanone monooxygenase (CPMO) from Comamonas (previously Pseudomonas) sp. NCI...

Baeyer-Villiger cyclohexanone 1,2-monooxygenase (CHMO) was purified 17.1-fold from cell extracts of the fungus Exophiala jeanselmei grown on cyclohexanol to electrophoretically homogeneity by serial chromatographies. The molecular mass of the native enzyme was approximately 74 kDa by gel filtration and SDS-PAGE. Some enzymic characterizations were studied. The NH2-terminal amino acid residues were Ala-Lys-Ser-Leu-Asp-Val-Leu-Ile-Val-Gly-Ala-Gly-Phe-Gly-Gly-Ile-Tyr-Gln-Leu-, with similarity to the bacterial CHMOs of FAD-binding and NADPH-dependent type Baeyer- Villiger monooxygenases.

Six cyclohexylacetic acid-degrading strains were isolated from soil samples in Japan and identified as members of the genera Cupriavidus (strain KUA-1), Rhodococcus, and Dietzia by 16S rRNA gene sequence analysis. For the first time members of these genera were shown to be capable of degrading cyclohexylacetic acid. A selected strain, KUA-1, which is the first reported Gram-negative organism capable of growth on cyclohexylacetic acid, was identified as a Cupriavidus metallidurans, based on morphologic and physiologic characteristics and its 16S rRNA gene sequence. Metabolite analysis by HPLC-MS indicated that 1-cyclohexenylacetic acid is an intermediate of cyclohexaneacetic acid metabolism in strain KUA-1.

Pseudomonas fluorescens strain KU-7 is a prototype microorganism that metabolizes 2-nitrobenzoate (2-NBA) via the formation of 3-hydroxyanthranilate (3-HAA), a known antioxidant and reductant. The initial two steps leading to the sequential formation of 2-hydroxy/aminobenzoate and 3-HAA are catalyzed by a NADPH-dependent 2-NBA nitroreductase (NbaA) and 2-hydroxylaminobenzoate mutase (NbaB), respectively. The 216-amino-acid protein NbaA is 78% identical to a plasmid-encoded hypothetical conserved protein of Polaromonas strain JS666; structurally, it belongs to the homodimeric NADH:flavin mononucleotide (FMN) oxidoreductase-like fold family. Structural modeling of complexes with the flavin, coenzyme, and substrate suggested specific residues contributing to the NbaA catalytic activity, assuming a ping-pong reaction mechanism. Mutational analysis supports the roles of Asn40, Asp76, and Glu113, which are predicted to form the binding site for a divalent metal ion implicated in FMN binding, and a role in NADPH binding for the 10-residue insertion in the β5-α2 loop. The 181-amino-acid sequence of NbaB is 35% identical to the 4-hydroxylaminobenzoate lyases (PnbBs) of various 4-nitrobenzoate-assimilating bacteria, e.g., Pseudomonas putida strain TW3. Coexpression of nbaB with nbaA in Escherichia coli produced a small amount of 3-HAA from 2-NBA, supporting the functionality of the nbaB gene. We also showed by gene knockout and chemotaxis assays that nbaY , a chemoreceptor NahY homolog located downstream of the nbaA gene, is responsible for strain KU-7 being attracted to 2-NBA. NbaY is the first chemoreceptor in nitroaromatic metabolism to be identified, and this study completes the gene elucidation of 2-NBA metabolism that is localized within a 24-kb chromosomal locus of strain KU-7.

Phenolic compounds are widely distributed toxic pollutants in seawater, and their effective degradation is very important for bioremediation programs. In this study, nine phenol-degrading bacteria were isolated from seawater samples, which were collected from the coastal areas of Japan. Besides the enrichment substrate phenol, all isolates could utilize at least one isomer of cresol as the sole source of carbon. A 16S rRNA gene sequence analysis indicated that all strains were affiliated with the class Gammaproteobacteria, four strains were closely related to Spongiibacter, four were closely related to Marinobacter, and one was closely related to Photobacterium. During growth on phenol, all isolates produced a yellow product, and a whole-cell study indicated that it was an extradiol meta-ring cleavage product of catechol, 2-hydroxymuconate semialdehyde. Phylogenetic analysis revealed that the partial gene encoding the largest subunit of the multicomponent phenol hydroxylase of the isolates was similar to that of terrestrial bacteria, thereby suggesting that phenol is converted into catechol by marine bacteria. We also suggest that horizontal transfer of the gene may occur not only among marine bacteria but also between the genera Marinobacter and Pseudomonas.

The enzymatic activity of Pseudomonas fluorescens alpha-amino-beta-carboxymuconic-epsilon-semialdehyde decarboxylase (ACMSD) is critically dependent on a transition metal ion [Li, T., Walker, A. L., Iwaki, H., Hasegawa, Y., and Liu, A. (2005) J. Am. Chem. Soc. 127, 12282-12290]. Sequence analysis in this study further suggests that ACMSD belongs to the amidohydrolase superfamily, whose structurally characterized members comprise a catalytically essential metal cofactor. To identify ACMSD's metal ligands and assess their functions in catalysis, a site-directed mutagenesis analysis was conducted. Alteration of His-9, His-177, and Asp-294 resulted in a dramatic loss of enzyme activity, substantial reduction of the metal-binding ability, and an altered metallocenter electronic structure. Thus, these residues are confirmed to be the endogenous metal ligands. His-11 is implicated in metal binding because of the strictly conserved HxH motif with His-9. Mutations at the 228 site yielded nearly inactive enzyme variants H228A and H228E. The two His-228 mutant proteins, however, exhibited full metal-binding ability and a metal center similar to that of the wild-type enzyme as shown by EPR spectroscopy. Kinetic analysis on the mutants indicates that His-228 is a critical catalytic residue along with the metal cofactor. Since the identified metal ligands and His-228 are present in all known ACMSD sequences, it is likely that ACMSD proteins from other organisms contain the same cofactor and share similar catalytic mechanisms. ACMSD is therefore the first characterized member in the amidohydrolase superfamily that represents a C-C breaking activity.

Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed in Escherichia coli a new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived from Pseudomonas sp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of ∼60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 μmol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains ∼1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme ( K m = 8 μM versus K m = 24 μM). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C 11 to C 15 ketones, methyl-substituted C 5 and C 6 ketones, and bicyclic ketones, such as decalone and β-tetralone. CPDMO has the highest affinity ( K m = 5.8 μM) and the highest catalytic efficiency ( k cat / K m ratio of 7.2 × 10 5 M −1 s −1 ) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity ( E > 200) as well as 99% S -selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although showing a modest selectivity ( E = 5.8), macrolactone formation of 15-hexadecanolide from the kinetic resolution of 2-methylcyclopentadecanone using CPDMO was also demonstrated.

The 2-nitrobenzoic acid degradation pathway of Pseudomonas fluorescens strain KU-7 proceeds via a novel 3-hydroxyanthranilate intermediate. In this study, we cloned and sequenced a 19-kb DNA locus of strain KU-7 that encompasses the 3-hydroxyanthranilate meta -cleavage pathway genes. The gene cluster, designated nbaEXHJIGFCDR, is organized tightly and in the same direction. The nbaC and nbaD gene products were found to be novel homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase, respectively. The NbaC enzyme carries out the oxidation of 3-hydroxyanthranilate to 2-amino-3-carboxymuconate-6-semialdehyde, while the NbaD enzyme catalyzes the decarboxylation of the latter compound to 2-aminomuconate-6-semialdehyde. The NbaC and NbaD proteins were overexpressed in Escherichia coli and characterized. The substrate specificity of the 23.8-kDa NbaC protein was found to be restricted to 3-hydroxyanthranilate. In E. coli , this enzyme oxidizes 3-hydroxyanthranilate with a specific activity of 8 U/mg of protein. Site-directed mutagenesis experiments revealed the essential role of two conserved histidine residues (His52 and His96) in the NbaC sequence. The NbaC activity is also dependent on the presence of Fe 2+ but is inhibited by other metal ions, such as Zn 2+ , Cu 2+ , and Cd 2+ . The NbaD protein was overproduced as a 38.7-kDa protein, and its specific activity towards 2-amino-3-carboxymuconate-6-semialdehyde was 195 U/mg of protein. Further processing of 2-aminomuconate-6-semialdehyde to pyruvic acid and acetyl coenzyme A was predicted to proceed via the activities of NbaE, NbaF, NbaG, NbaH, NbaI, and NbaJ. The predicted amino acid sequences of these proteins are highly homologous to those of the corresponding proteins involved in the metabolism of 2-aminophenol (e.g., AmnCDEFGH in Pseudomonas sp. strain AP-3). The NbaR-encoding gene is predicted to have a regulatory function of the LysR family type. The function of the product of the small open reading frame, NbaX, like the homologous sequences in the nitrobenzene or 2-aminophenol metabolic pathway, remains elusive.