The antibiotic roseoflavin from Streptomyces davawensis:
Mechanism of action, resistance and biosynthesis

Roseoflavin (RoF) produced by Streptomyces davawensis and Streptomyces cinnabarinus is the only known natural riboflavin (RF) analog with antibiotic function and is studied as a model compound in our laboratory. RoF is taken up by RF transporters and is converted to the FMN/FAD analogs RoFMN/RoFAD by flavokinases/FAD-synthetases. The addition of RoF leads to a decrease of intracellular FMN/FAD levels. This is the consequence of RoFMN mediated blocking of FMN riboswitches, which leads to a reduced expression of genes involved in RF transport and/or biosynthesis. This reduced supply with RF is thought to be one of the reasons for RoF toxicity. Notably, a specialized FMN riboswitch confers roseoflavin resistance to S. davawensis and S. cinnabarinus. Additional cellular targets for RoF are flavoproteins. It was found that 37 out of 38 Escherichia coli flavoproteins contained either RoFMN or RoFAD when cells were treated with just toxic doses of RoF. FMN-dependent AzoR (EC 1.7.1.6) from E. coli exemplarily was analyzed in greater detail with regard to the molecular effect of RoF. RoFMN binds to AzoR apoenzyme with a higher affinity compared to that of FMN, however, AzoR-RoFMN is less acitve (30% of AzoR-FMN activity). Structural analysis (1.07 Å) revealed that RoFMN binding did not affect the overall topology of the enzyme and also did not interfere with dimerization of AzoR.

It was postulated that RoF is synthesized from RF through 8-amino-8-demethyl-riboflavin (AF) and 8-methylamino-8-demethyl-riboflavin (MAF). We could identify a N,N-dimethyltransferase (RosA) catalyzing the two sequential SAM-dependent methylation reactions necessary to convert AF into RoF. We have sequenced the genome of S. davawensis (9,466,619-bp linear chromosome/89,331-bp linear plasmid). Heterologous expression of a set of genes present on a 106 kbp subgenomic fragment of S. davawensis resulted in the production of roseoflavin by the host Streptomyces coelicolor M1152. These genes (present on cosmid pESAC-13) were systematically replaced by resistance cassettes (“knock-out”). This stepwise approach and additional gene expression experiments suggest that the conversion of RF to AF is catalyzed by a single enzyme. This enzyme has been purified to apparent homogeneity and currently is characterized in our laboratory.