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 flavindependent 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.

Mechanism of 4-Nitrophenol Oxidation in Rhodococcus sp. Strain PN1: Characterization of the Two-Component 4-Nitrophenol Hydroxylase and Regulation of Its Expression

“…The nph and npc gene clusters include an AraC-type regulatory gene (nphR) and a LysR-type regulatory gene (ORF1), respectively, while the npd gene cluster includes a large MalT-DnrI-fused-type regulatory gene (npdR). Although the npc and npd gene clusters were transcribed in the cells grown on 4-NP (22,29), little is known about the regulatory systems for their expression, including the functions of ORF1 and npdR. We demonstrated in the present study that nphR encodes a positive regulatory protein for the expression of nphA1 and that 4-NP is the only molecule capable of functioning as an inducer (of the several compounds tested).…”

“…strain PN1, R. opacus SAO101, and Arthrobacter sp. strain JS443 (22,29,43). The genetic organizations of these clusters are quite different in each microorganism.…”

“…Very recently, Perry and Zylstra cloned a 4-NP monooxygenase gene cluster (npdBA1RXA2C) from Arthrobacter sp. strain JS443 (29) that showed significant homology with the chlorophenol 4-monooxygenase gene cluster of Arthrobacter chlorophenolicus A6 (27). In this cluster, a flavin reductase gene (npdA1) and a monooxygenase gene (npdA2) are separated by a large putative regulatory gene (npdR) and transcribed divergently.…”

Identification and Characterization of Catabolicpara-Nitrophenol 4-Monooxygenase andpara-Benzoquinone Reductase fromPseudomonassp. Strain WBC-3

“…4). The detection of hydroquinone may be due to benzoquinone reduction in the presence NADPH, and the same explanation was proposed previously for strain JS443 (25). In a time course assay of the monooxygenation reaction, the PNP consumption (75.6 M) was equivalent to the total accumulation of both benzoquinone (59.2 M) and hydroquinone (15.5 M) (Fig.…”

“…Another two-component PNP monooxygenase, NpdA1A2 from Arthrobacter sp. strain JS443, was found to be involved in the conversion of PNP to hydroxyquinol, but it does so via hydroxy-1,4-benzoquinone (25). More recently, a twocomponent PNP hydroxylase from Rhodococcus sp.…”