Identification and characterization of a bacterial cytochrome P450 monooxygenase catalyzing the 3-nitration of tyrosine in rufomycin biosynthesis.

Rufomycin is a circular heptapeptide with anti-mycobacterial activity and is produced by ATCC 14046. Its structure contains three non-proteinogenic amino acids, -dimethylallyltryptophan, -2-crotylglycine, and 3-nitrotyrosine (3NTyr). Although the rufomycin structure was already reported in the 1960s, its biosynthesis, including 3NTyr generation, remains unclear. To elucidate the rufomycin biosynthetic pathway, we assembled a draft genome sequence of and identified the rufomycin biosynthetic gene cluster ( cluster), consisting of 20 ORFs (). We found a putative heptamodular nonribosomal peptide synthetase encoded by , a putative tryptophan -dimethylallyltransferase encoded by , and a putative trimodular type I polyketide synthase encoded by Moreover, the cluster contains an apparent operon harboring putative cytochrome P450 () and nitric oxide synthase () genes. A similar operon, , is responsible for the formation of 4-nitrotryptophan in thaxtomin biosynthesis; the cytochrome P450 TxtE catalyzes the 4-nitration of Trp. Therefore, we hypothesized that RufO should catalyze the Tyr 3-nitration. Disruption of abolished rufomycin production by , which was restored when 3NTyr was added to the culture medium of the disruptant. Recombinant RufO protein exhibited Tyr 3-nitration activity both and Spectroscopic analysis further revealed that RufO recognizes Tyr as the substrate with a dissociation constant of ∼0.1 μm These results indicate that RufO is an unprecedented cytochrome P450 that catalyzes Tyr nitration. Taken together with the results of an analysis of the cluster, we propose a rufomycin biosynthetic pathway in .