Expression and characterization of an N-oxygenase from Rhodococcus jostii RHAI

The Journal of General and Applied Microbiology

Jed O. Eberly¹  Karl J. Indest¹  Dawn E. Hancock¹ 

[1] U.S. Army Engineer Research and Development Center, Environmental Laboratory

关键词: aromatic amino acids;    N-oxygenase;    Rhodococcus;   

DOI  :  10.2323/jgam.61.217

学科分类：微生物学和免疫学

来源: Applied Microbiology, Molecular and Cellulrar Biosciences Research Foundation

【 摘 要 】

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%.

【 授权许可】

Unknown