A tamed killer enzyme: In-depth analysis of the roseoflavin pathway specific phosphatase RosC from Streptomyces davaonensis

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Streptomyces davaonensis and Streptomyces cinnabarinus are the only known bacteria which synthesize the antibiotic roseoflavin and therefore are the only known organisms to contain roseoflavin biosynthetic enzymes and the unique roseoflavin pathway specific phosphatase RosC. Gene expression and gene deletion experiments carried out within previous project led to the identification of this last enzyme of the roseoflavin biosynthetic pathway. In cooperation with Ulrich Ermler (Max-Planck-Institute for biophysics, Frankfurt, Germany) the crystal structure of this novel protein was solved (up to 1.6 Å). Based on this work and based on preliminary site-directed mutagenesis experiments we tentatively identified specific amino acids in the active site of RosC which probably play an important role in substrate binding and catalysis. We now plan to validate these findings by additional site-directed mutagenesis experiments. Most importantly kinetic studies with a whole variety of different RosC mutants were not yet carried out. In contrast to all other members of the histidine phosphatase family RosC contains a 22 amino acid N-terminus. These amino acids of RosC form a unique alpha-helix which we hypothesize to function as a specialized “lid” contributing to substrate binding. This aspect shall now also be studied in the present follow-up application. We found that RosC also dephosphorylates FMN (flavin mononucleotide or riboflavin-5’-phosphate). FMN is one of the most abundant cofactors in all life forms. FMN is synthesized from riboflavin and ATP by the bifunctional S. davaonensis flavokinase/FAD synthetase RibCF which was also characterized in the Mack laboratory. Synthesis of the roseoflavin precursor FMN from riboflavin and ATP by RibCF and concomitant dephosphorylation of FMN by RosC would generate a futile cycle in roseoflavin producers. Thus, an important question regarding RosC is how the enzyme discriminates between the substrate AFP and the important cellular cofactor FMN. Structural data strongly suggest that D166 plays a key role and therefore our planned analyses will be centered (but not limited) to this residue. Our preliminary site-directed mutagenesis experiments within previous project revealed that overproduction of the RosC mutants RosC D166L, D166V and D166I in E. coli leads to reduced growth when synthesis of the recombinant protein is induced and suggests that RosC has a “potential” to dephosphorylate an important cellular phosphometabolite (or several). Notably, our in vitro data clearly show that it is neither FMN nor FAD which are dephosphorylated by these mutants. It is planned to identify this unknown phosphometabolite which possibly represents a novel target for antibiotic treatment in general as ist dephosphorylation is lethal for the cell.