Cloning of a gene cluster involved in the catabolism of p-nitrophenol by Arthrobacter sp. strain JS443 and characterization of the p-nitrophenol monooxygenase

Abstract

The npd gene cluster, which encodes the enzymes of a p-nitrophenol catabolic pathway from Arthrobacter sp. strain JS443, was cloned and sequenced. Three genes, npdB, npdA1, and npdA2, were independently expressed in Escherichia coli in order to confirm the identities of their gene products. NpdA2 is a p-nitrophenol monooxygenase belonging to the two-component flavin-diffusible monooxygenase family of reduced flavin-dependent monooxygenases. NpdA1 is an NADH-dependent flavin reductase, and NpdB is a hydroxyquinol 1,2-dioxygenase. The npd gene cluster also includes a putative maleylacetate reductase gene, npdC. In an in vitro assay containing NpdA2, an E. coli lysate transforms p-nitrophenol stoichiometrically to hydroquinone and hydroxyquinol. It was concluded that the p-nitrophenol catabolic pathway in JS443 most likely begins with a two-step transformation of p-nitrophenol to hydroxy-1,4-benzoquinone, catalyzed by NpdA2. Hydroxy-1,4-benzoquinone is reduced to hydroxyquinol, which is degraded through the hydroxyquinol ortho cleavage pathway. The hydroquinone detected in vitro is a dead-end product most likely resulting from chemical or enzymatic reduction of the hypothetical intermediate 1,4-benzoquinone. NpdA2 hydroxylates a broad range of chloro- and nitro-substituted phenols, resorcinols, and catechols. Only p-nitro- or p-chloro-substituted phenols are hydroxylated twice. Other substrates are hydroxylated once, always at a position para to a hydroxyl group.

FIG. 1.

Proposed pathway for degradation of PNP by Arthrobacter sp. strain JS443. Hypothetical intermediates are indicated by brackets. The pathway encoded by the npd gene cluster proceeds through 1,4-benzoquinone and hydroxy-1,4-benzoquinone, as shown in this study. In strain JS443, an unknown oxygenase diverts an unknown proportion of PNP to 4-nitrocatehcol (20), which reenters the npd pathway after being oxidized by NpdA2. Accumulation of hydroquinone (presumably through reduction of 1,4-benzoquinone) was observed by Jain et al. (20) and in the current study, but its fate is unknown. Hydroquinone is not metabolized by either NpdA2 or NpdB. FADH₂, reduced flavin adenine dinucleotide.

FIG. 2.

Organization of (A) the npd gene cluster from Arthrobacter sp. strain JS443 and (B) the homologous cph gene cluster which encodes 4-chlorophenol catabolism in A. chlorophenolicus A6 (accession number AAN08754) (25). cphR and cphS are putative regulatory genes which are homologous to the 5′ and 3′ and, respectively, of npdR. Putative functions are shown below the cph genes. Abbreviations: f-mono, flavoprotein monooxygenase; mar, maleylacetate reductase; hql, hydroxyquinol 1,2-dioxygenase; res, resolvase pseudogene; f-red, flavin reductase; reg, transcriptional regulator; t-mono, TC-FDM.

FIG. 3.

Transformation of PNP by an E. coli lysate containing NpdA2. The rates of substrate disappearance (PNP [▪]) and product appearance (hydroxyquinol [•] and hydroquinone [▴]) are averages from at least three assays, carried out using independent preparations of E. coli BL21(DE3)(pLysS, pLP261), as described in Materials and Methods. Samples were withdrawn at the indicated time points and treated immediately with glacial acetic acid (1%) and sodium dithionite (1 mM) to stop the reaction and reduce quinones to quinols. Substrates and products were quantified by HPLC.

FIG. 4.

Mass spectra of the acetylated derivatives of the NpdA2 transformation product identified as 5-chlorohydroxyquinol. Transformations were carried out using cleared lysates of E. coli BL21(DE3)(pLysS, pLP261) as the source of NpdA2, as described in Materials and Methods. The transformation substrates were 2,4,5-trichlorophenol (A) and 4-chlorocatechol (B). Following incubation for 30 min at 25°C, reaction mixtures were acetylated with acetic anhydride and analyzed by GC-MS.

FIG. 5.

Mass spectra of the acetylated derivatives of 2,4,6-trichlorophenol transformation products. (A) Product identified as 2,6-dichlorohydroquinone. (B) Product identified as 6-chlorohydroxyquinol. (C and D) Unidentified transformation products. Transformations were carried out using cleared lysates of E. coli BL21(DE3)(pLysS, pLP261) as the source of NpdA2, as described in Materials and Methods. Following incubation for 30 min at 25°C, reaction mixtures were acetylated with acetic anhydride and analyzed by GC-MS.

FIG. 6.

Mass spectra of the p-cresol transformation product identified as HMCH (A) and authentic HMCH (B). Transformations were carried out using cleared lysates of E. coli BL21(DE3)(pLysS, pLP261) as the source of NpdA2, as described in Materials and Methods.

FIG. 7.

Fate of hypothetical monooxygenase reaction intermediates. (A) Monooxygenase attack at a site occupied by an electron-withdrawing substituent. (B) Monooxygenase attack at a site occupied by an electron-donating substituent. (C) Transformation of p-cresol to HMCH by NpdA2.