title: Expression and characterization of an N-oxygenase from Rhodococcus jostii RHAI.
abstract: Nitro group-containing natural products are rare in nature. There are few examples of N-oxygenases, enzymes that incorporate atmospheric oxygen into primary and secondary amines, characterized in the literature. N-oxygenases have yet to be characterized from the Corynebacterineae, a metabolically diverse group of organisms that includes the genera Rhodococcus, Gordonia, and Mycobacterium. A preliminary in silico search for N-oxygenase AurF gene orthologs revealed multiple protein candidates present in the genome of the Actinomycete Rhodococcus jostii RHAI (RHAI_ro06104). Towards the goal of identifying novel biocatalysts with potential utility for the biosynthesis of nitroaromatics, AurF ortholog RHAI_ro6104 was cloned, expressed and purified in E. coli and amine and nitro containing phenol substrates tested for activity. RHAI-ro06104 showed the highest activity with 4-aminophenol, producing a Vmax of 18.76 μM s(-1) and a Km of 15.29 mM and demonstrated significant activities with 2-aminophenol and 2-amino-5-methylphenol, producing a Vmax of 12.86 and 12.72 μM s(-1) with a Km of 8.34 and 2.81 mM, respectively. These findings are consistent with a substrate range observed in other N-oxygenases, which seem to accommodate substrates that lack halogenated substitutions and side groups directly flanking the amine group. Attempts to identify modulators of RHAI-ro06104 gene activity demonstrated that aromatic amino acids inhibit expression by almost 50%.
authors.affiliation: U.S. Army Engineer Research and Development Center, Environmental Laboratory.
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Indest
authors.forename: Jed O
authors.initials: JO
authors.lastname: Eberly
authors.forename: Dawn E
authors.initials: DE
authors.lastname: Hancock
mesh_terms: D000627:Aminophenols / Q000378:metabolism*; D003001:Cloning, Molecular; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism*; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D000627:Aminophenols; D009596:Nitrophenols; D010105:Oxygenases