Identification and Characterization of Catabolic para-Nitrophenol 4-Monooxygenase and para-Benzoquinone Reductase from Pseudomonas sp Strain WBC-3

Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, we cloned and sequenced a 12.7-kb fragment containing a conserved region of NAD(P) H: quinone oxidoreductase genes. Of the products of the 13 open reading frames deduced from this fragment, PnpA shares 24% identity to the large component of a 3-hydroxy-phenylacetate hydroxylase from Pseudomonas putida U and PnpB is 58% identical to an NAD(P) H: quinone oxidoreductase from Escherichia coli. Both PnpA and PnpB were purified to homogeneity as His-tagged proteins, and they were considered to be a monomer and a dimer, respectively, as determined by gel filtration. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase that converts PNP to para-benzoquinone in the presence of NADPH. PnpB is a flavin mononucleotide-and NADPH-dependent p-benzoquinone reductase that catalyzes the reduction of p-benzoquinone to hydroquinone. PnpB could enhance PnpA activity, and genetic analyses indicated that both pnpA and pnpB play essential roles in PNP mineralization in strain WBC-3. Furthermore, the pnpCDEF gene cluster next to pnpAB shares significant similarities with and has the same organization as a gene cluster responsible for hydroquinone degradation (hapCDEF) in Pseudomonas fluorescens ACB (M.J. Moonen, N.M. Kamerbeek, A.H. Westphal, S.A. Boeren, D.B. Janssen, M.W. Fraaije, and W.J. van Berkel, J. Bacteriol. 190: 5190-5198, 2008), suggesting that the genes involved in PNP degradation are physically linked.