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

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.