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

达文链霉菌和朱砂链霉菌是唯一已知的合成抗生素玫瑰黄素的细菌，因此是唯一已知的含有玫瑰黄素生物合成酶和独特的玫瑰黄素途径特异性磷酸酶RosC的生物体。在先前的项目中进行的基因表达和基因缺失实验导致了玫瑰黄素生物合成途径的最后一种酶的鉴定。在与Ulrich Ermler（Max-Planck-Institute for biophysics，法兰克福，德国）的合作下，解决了这种新型蛋白质的晶体结构（高达1.6 μ m）。在此基础上，初步的定点突变实验的基础上，我们初步确定了特定的氨基酸在活性位点的RosC可能发挥重要作用的底物结合和催化。我们现在计划通过额外的定点诱变实验来验证这些发现。最重要的是，还没有对各种不同的RosC突变体进行动力学研究。与组氨酸磷酸酶家族的所有其他成员相比，RosC含有22个氨基酸的N-末端。RosC的这些氨基酸形成独特的α-螺旋，我们假设其充当有助于底物结合的专门的"盖"。这方面现在也将在本后续申请中进行研究。我们发现，RosC也使FMN（黄素单胞苷或核黄素-5'-磷酸）去磷酸化。FMN是所有生命形式中最丰富的辅因子之一。FMN是由核黄素和ATP通过双功能S. davaonensis黄素激酶/FAD合成酶RibCF，其也在Mack实验室中表征。通过RibCF从核黄素和ATP合成玫瑰黄素前体FMN以及伴随的通过RosC的FMN的去磷酸化将在玫瑰黄素生产者中产生无效循环。因此，关于RosC的一个重要问题是酶如何区分底物AFP和重要的细胞辅因子FMN。结构数据强烈表明D166起关键作用，因此我们计划的分析将集中（但不限于）于该残留物。我们在前期项目中进行的初步定点突变实验表明，RosC突变体RosC D166L、D166V和D166I在E.当重组蛋白的合成被诱导时，大肠杆菌的RosC导致生长减少，这表明RosC具有使重要的细胞磷酸代谢物（或几种）去磷酸化的"潜力"。值得注意的是，我们的体外数据清楚地表明，FMN和FAD都不会被这些突变体去磷酸化。计划鉴定这种未知的磷酸代谢物，其可能代表抗生素治疗的新靶点，因为其去磷酸化对细胞是致命的。

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.