Molecular and biochemical analysis of p-nitrophenol degradation by Arthrobacter sp. strain JS443

The p-nitrophenol degradation pathway of Arthrobacter sp. strain JS443 is initiated by oxidation of p-nitrophenol to hydroxyquinol (1,2,4-trihydroxybenzene). Hydroxyquinol is cleaved by hydroxyquinol 1,2-dioxygenase to yield maleylacetate, which enters the TCA cycle through the beta-ketoadipate pathway. Hydroxyquinol 1,2-dioxygenase was purified and its substrate range, kinetic parameters, and N-terminal amino acid sequence were determined. In order to facilitate characterization of other enzymes involved in this pathway, the genes encoding the pathway were cloned using a DNA probe derived from the N-terminal sequence of hydroxyquinol 1,2-dioxygenase. A total of 12,559 by of DNA was cloned and sequenced. This DNA sequence contains a 912 base pair open reading frame, npdB, which encodes a protein with an N-terminal sequence identical to that of hydroxyquinol 1,2-dioxygenase. Genes homologous to flavin reductases, reduced flavin-dependant monooxygenases, maleylacetate reductases, and transcriptional regulators were also found. Three genes, npdB, npdA1, and npdA2, were expressed in E. coli in order to confirm the identities of their gene products. NpdA1, which was purified as a His-tagged fusion protein, was found to be a flavin reductase with a significant preference for FAD as the flavin substrate. An in vitro assay was developed to investigate the substrate range and cofactor requirements of NpdA2, the putative monooxygenase. The assay contained purified NpdA1-His, crude lysate from an E. coli strain expressing NpdA2, FAD, NAD, and an NADH-regeneration system. FADH2, the probable electron donor for NpdA2, could not be added directly to the reaction due to its instability in the presence of oxygen. NpdA2 was shown to hydroxylate a broad range of chloro- and nitro-substituted phenols, resorcinols, and catechols. p-Nitrophenol and other p-chloro- and p-nitro-substituted phenols are hydroxylated twice by NpdA2. Other substrates are hydroxylated only once. Substrates are hydroxylated in a regioselective manner. NpdA2 activity is significantly diminished when NpdA1 or FAD is omitted from the reaction or when FAD is substituted with FMN.