The Phosphatase RosC from Streptomyces davaonensis is Used for Roseoflavin Biosynthesis and has Evolved to Largely Prevent Dephosphorylation of the Important Cofactor Riboflavin-5'-phosphate.

The antibiotic roseoflavin is a riboflavin (vitamin B2) analog. One step of the roseoflavin biosynthetic pathway is catalyzed by the phosphatase RosC, which dephosphorylates 8-demethyl-8-amino-riboflavin-5'-phosphate (AFP) to 8-demethyl-8-amino-riboflavin (AF). RosC also catalyzes the potentially cell-damaging dephosphorylation of the AFP analog riboflavin-5'-phosphate also called "flavin mononucleotide" (FMN), however, with a lower efficiency. We performed X-ray structural analyses and mutagenesis studies on RosC from Streptomyces davaonensis to understand binding of the flavin substrates, the distinction between AFP and FMN and the catalytic mechanism of this enzyme. This work is the first structural analysis of an AFP phosphatase. Each monomer of the RosC dimer consists of an α/ß-fold core, which is extended by three specific elongated strand-to-helix sections and a specific N-terminal helix. Altogether these segments envelope the flavin thereby forming a novel flavin-binding site. We propose that distinction between AFP and FMN is provided by substrate-induced rigidification of the four RosC specific supplementary segments mentioned above and by an interaction between the amino group at C8 of AFP and the ß-carboxylate of D166. This key amino acid is involved in binding the ring system of AFP and positioning its ribitol phosphate part. Accordingly, site-specific exchanges at D166 disturbed the active site geometry of the enzyme and drastically reduced the catalytic activity. Based on the structure of the catalytic core we constructed a whole series of RosC variants but a disturbing, FMN dephosphorylating "killer enzyme", was not generated.

A second riboflavin import system is present in flavinogenic Streptomyces davaonensis and supports roseoflavin biosynthesis.

The antibiotic roseoflavin is produced by Streptomyces davaonensis in the stationary phase of growth. To support biosynthesis of the secondary metabolite roseoflavin, S. davaonensis underwent several genetic adaptations with regard to metabolism of the roseoflavin precursor and primary metabolite riboflavin. In addition to 17 riboflavin biosynthesis genes at different chromosomal locations, S. davaonensis contains the riboflavin transporter gene ribM being part of the riboflavin biosynthetic operon ribE1MAB5H. Deletion of this operon generated riboflavin auxotrophic S. davaonensis strains. The finding that S. davaonensis ΔribE1MAB5H was able to grow in a culture medium containing low levels of riboflavin indicated that in addition to RibM, a second riboflavin transporter is present in this bacterium. The S. davaonensis genes ribXY (former rosXY) represented candidate genes for such a second riboflavin transport system and the results of our experiments now show that RibXY from S. davaonensis is a highly efficient riboflavin importer but not a roseoflavin importer.

The novel phosphatase RosC catalyzes the last unknown step of roseoflavin biosynthesis in Streptomyces davaonensis.

The bacterium Streptomyces davaonensis produces the antibiotic roseoflavin, which is a riboflavin (vitamin B2 ) analog. The key enzyme of roseoflavin biosynthesis is the 8-demethyl-8-amino-riboflavin-5'-phosphate (AFP) synthase RosB which synthesizes AFP from riboflavin-5'-phosphate. AFP is not a substrate for the last enzyme of roseoflavin biosynthesis the N, N-dimethyltransferase RosA, which generates roseoflavin from 8-demethyl-8-amino-riboflavin (AF). Consequently, the roseoflavin biosynthetic pathway depends on a phosphatase, which dephosphorylates AFP to AF. Here, we report on the identification and characterization of such an AFP phosphatase which we named RosC. The gene rosC is located immediately downstream of rosA and both genes are part of a cluster comprising 10 genes. Deletion of rosC from the chromosome of S. davaonensis led to reduced roseoflavin levels in the corresponding recombinant strain. In contrast to wild-type S. davaonensis, cell-free extracts of the rosC deletion strain did not catalyze dephosphorylation of AFP. RosC was purified from an overproducing Escherichia coli strain. RosC is the fastest enzyme of roseoflavin biosynthesis (kcat 31.3 ± 1.4 min-1 ). The apparent KM for the substrate AFP was 34.5 µM. Roseoflavin biosynthesis is now completely understood--it takes three enzymes (RosB, RosC, and RosA) to convert the flavin cofactor riboflavin-5'-phosphate into a potent antibiotic.

Metabolic engineering of roseoflavin-overproducing microorganisms.

BACKGROUND: Roseoflavin, a promising broad-spectrum antibiotic, is naturally produced by the bacteria Streptomyces davaonensis and Streptomyces cinnabarinus. The key enzymes responsible for roseoflavin biosynthesis and the corresponding genes were recently identified. In this study we aimed to enhance roseoflavin production in S. davaonensis and to synthesize roseoflavin in the heterologous hosts Bacillus subtilis and Corynebacterium glutamicum by (over)expression of the roseoflavin biosynthesis genes. RESULTS: While expression of the roseoflavin biosynthesis genes from S. davaonensis was not observed in recombinant strains of B. subtilis, overexpression was successful in C. glutamicum and S. davaonensis. Under the culture conditions tested, a maximum of 1.6 ± 0.2 µM (ca. 0.7 mg/l) and 34.9 ± 5.2 µM (ca. 14 mg/l) roseoflavin was produced with recombinant strains of C. glutamicum and S. davaonensis, respectively. In S. davaonensis the roseoflavin yield was increased by 78%. CONCLUSIONS: The results of this study provide a sound basis for the development of an economical roseoflavin production process.

Comparative biochemical and structural analysis of the flavin-binding dodecins from Streptomyces davaonensis and Streptomyces coelicolor reveals striking differences with regard to multimerization.

Dodecins are small flavin-binding proteins that are widespread amongst haloarchaeal and bacterial species. Haloarchaeal dodecins predominantly bind riboflavin, while bacterial dodecins have been reported to bind riboflavin-5'-phosphate, also called flavin mononucleotide (FMN), and the FMN derivative, flavin adenine dinucleotide (FAD). Dodecins form dodecameric complexes and represent buffer systems for cytoplasmic flavins. In this study, dodecins of the bacteria Streptomyces davaonensis (SdDod) and Streptomyces coelicolor (ScDod) were investigated. Both dodecins showed an unprecedented low affinity for riboflavin, FMN and FAD when compared to other bacterial dodecins. Significant binding of FMN and FAD occurred at relatively low temperatures and under acidic conditions. X-ray diffraction analyses of SdDod and ScDod revealed that the structures of both Streptomyces dodecins are highly similar, which explains their similar binding properties for FMN and FAD. In contrast, SdDod and ScDod showed very different properties with regard to the stability of their dodecameric complexes. Site-directed mutagenesis experiments revealed that a specific salt bridge (D10-K62) is responsible for this difference in stability.

Taxonomic analyses of members of the Streptomyces cinnabarinus cluster, description of Streptomyces cinnabarigriseus sp. nov. and Streptomyces davaonensis sp. nov.

Roseoflavin is the only known riboflavin (vitamin B2) analog with antibiotic properties. It is actively taken up by many micro-organisms and targets flavinmononucleotide riboswitches and flavoproteins. It is described as the product of the tentatively named 'Streptomyces davawensis' JCM 4913. Taxonomic analysis of this strain with a polyphasic approach showed that it is very closely related to Streptomyces cinnabarinus (DSM 40467). The two Streptomyces isolates were obtained from different geographical locations (the Philippines and the Kamchatka Peninsula, respectively), their genomes have been sequenced and the question was whether or not the two isolates were representatives of the same species. As we also worked with another isolate of Streptomyces cinnabarinus JS 360, the producer of the cinnabaramides, we wanted to clarify the taxonomic position of the three isolates by using a polyphasic approach. After analysis of the 16S rRNA gene sequence, we found in total 23 species of the genus Streptomyces that showed a similarity higher than 98.5 % to the three strains. We showed that 'S. davawensis' JCM 4913 and S. cinnabarinus DSM 40467 were very closely related but belong to two different species. Hence, we validate 'S. davawensis' as Streptomyces davaonensis sp. nov. with the type strain JCM 4913T (=DSM 101723T). In addition, the cinnabaramide producer can be clearly differentiated from S. davaonensis and this isolate is described as Streptomyces cinnabarigriseus sp. nov. with strain JS360T (=NCCB 100590T=DSM 101724T) as the type strain.