Cezomycin is activated by CalC to its ester form for further biosynthesis steps in the production of calcimycin in Streptomyces chartreusis NRRL 3882

Calcimycin, N-demethyl calcimycin and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis . A thorough understanding of the organization of their encoding genes, biosynthetic pathway (s) and cation specificities is vitally important for their efficient future production and therapeutic use. So far this has been lacking, as well as information concerning any biosynthetic relationships which may exist between calcimycin and cezomycin. In this study we observed that when a Cal - ( calB1 mutant) derivative of a calcimycin producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, calcimycin production could be restored. This suggested that calcimycin synthesis may have resulted from post-synthetic modification of cezomycin rather than de novo through a novel and independent biosynthetic mechanism. Systematic screening of a number Cal - S. chartreusis mutants lacking the ability to convert cezomycin to calcimycin allowed the identification of a gene, provisionally named calC , which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating CoA with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein9s apparent K m and k cat for cezomycin were observed to 190 μM and 3.98 min -1 respectively and it possessed oligomeric form in solution. Our results unequivocally show that cezomycin is post synthetically modified to calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form. Importance Calcimycin is a secondary metabolite divalent cation-ionophore which has been studied in the context of human health. However, detail is lacking with respect to both calcimycin9s biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues, divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how calcimycin and related compounds may be effective in modifying calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of calcimycin, which is essentially a derivative of cezomycin and allow us to propose a biosynthetic mechanism for calcimycin9s production.