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J. Bacteriol. doi:10.1128/JB.01849-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Cloning of a Gene Cluster Involved in the Catabolism of p-Nitrophenol by Arthrobacter sp. Strain JS443 and Characterization of the p-Nitrophenol Monooxygenase

Biotechnology Center for Agriculture and the Environment, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901

^* To whom correspondence should be addressed. Email: llperry{at}purdue.edu.

	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 E. coli in order to confirm the identities of their gene products. NpdA2 is a p-nitrophenol monooxygenase belonging to the TC-FDM 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. An in vitro assay containing NpdA2 in 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.