Roseoflavin is a bright red-colored anti-vitamin biosynthesized by Streptomyces davawensis and Streptomyces cinnabarinus. It is the only naturally occurring riboflavin analog that exhibits antibiotic properties. Earlier studies show that roseoflavin is biosynthesized from riboflavin by substitution of the C8 methyl with a dimethylamino group1. Recently, the methyltransferase catalyzing the final step has been characterized and a sub-genomic fragment containing genes sufficient for the entire biosynthesis has also been cloned. However, the mechanism of biosynthesis of the key intermediate 8-amino-flavin is poorly understood. Herein, we report the identification and characterization of a single flavin-dependent enzyme, RosB that replaces the C8 methyl group of Flavin mononucleotide (FMN) and uses the amino group of glutamate to convert FMN to 8-amino-FMN via the intermediacy of 8-formyl-FMN. The enzyme was cloned, overexpressed and purified, and subsequently tested for the predicted activity. A novel mechanism for the RosB-catalyzed reaction is proposed based on the identification of reaction products and intermediates. In the first step of this reaction, the enzyme oxidizes the C8 methyl group of FMN to a formyl group using molecular oxygen. Next, the intermediate, 8-formyl-FMN catalyzes the transfer of the amino group from the glutamate to flavin. The resulting reactive FMN intermediate then rearranges to yield 8-amino-FMN and the C8 methyl group is lost as formate. To gain further insights into this unprecedented amine transfer reaction various substrate analogs were designed and studied. Reactive intermediates were successfully trapped and characterized by HPLC and LCMS studies. Additionally, the crystal structure of the enzyme containing a bound inhibitor complemented the results of the biochemical analysis. Overall, this work reveals the existence of yet another novel flavin transformation involved in natural product biosynthesis. Moreover, we identified and characterized the phosphatase, RosC that converts 8-amino FMN, the product of RosB to 8-amino riboflavin, the substrate of the next enzyme in roseoflavin biosynthesis pathway. Thus, identification of RosB and RosC completed the biosynthetic pathway of roseoflavin. These studies demonstrated the power of mechanism guided approach to identify enzymes involved in natural product biosynthesis pathway.
