title: Characterization of a pseudomonad 2-nitrobenzoate nitroreductase and its catabolic pathway-associated 2-hydroxylaminobenzoate mutase and a chemoreceptor involved in 2-nitrobenzoate chemotaxis.
abstract: 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 beta5-alpha2 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.
authors.affiliation: Department of Biotechnology, Faculty of Engineering and High Technology Research Center, Kansai University, Suita, Osaka 564-8680, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Iwaki
authors.forename: Takamichi
authors.initials: T
authors.lastname: Muraki
authors.forename: Shun
authors.initials: S
authors.lastname: Ishihara
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: Kathryn N
authors.initials: KN
authors.lastname: Rankin
authors.forename: Traian
authors.initials: T
authors.lastname: Sulea
authors.forename: Jason
authors.initials: J
authors.lastname: Boyd
authors.forename: Peter C K
authors.initials: PC
authors.lastname: Lau
mesh_terms: D015095:3-Hydroxyanthranilic Acid / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D062365:Aminobenzoates / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002633:Chemotaxis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009579:Nitrobenzoates / Q000378:metabolism*; D009601:Nitroreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D016958:Pseudomonas putida / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: C518437:2-nitrobenzoate; D062365:Aminobenzoates; D009579:Nitrobenzoates; D015095:3-Hydroxyanthranilic Acid; D009601:Nitroreductases; D019751:Intramolecular Transferases