Characterization of a Pseudomonad 2-Nitrobenzoate Nitroreductase and Its Catabolic Pathway-Associated 2-Hydroxylaminobenzoate Mutase and a Chemoreceptor Involved in 2-Nitrobenzoate Chemotaxis

<jats:title>ABSTRACT</jats:title> <jats:p> <jats:italic>Pseudomonas fluorescens</jats:italic> 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 <jats:italic>Polaromonas</jats:italic> 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., <jats:italic>Pseudomonas putida</jats:italic> strain TW3. Coexpression of <jats:italic>nbaB</jats:italic> with <jats:italic>nbaA</jats:italic> in <jats:italic>Escherichia coli</jats:italic> produced a small amount of 3-HAA from 2-NBA, supporting the functionality of the <jats:italic>nbaB</jats:italic> gene. We also showed by gene knockout and chemotaxis assays that <jats:italic>nbaY</jats:italic> , a chemoreceptor NahY homolog located downstream of the <jats:italic>nbaA</jats:italic> 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. </jats:p>