Oxidative cleavage of premithramycin B is one of the last steps in the biosynthesis of the antitumor drug mithramycin

Background: Mithramycin is a member of the clinically important aureolic ac id group of antitumor drugs that interact with GO-rich regions of DNA nonin tercalatively. These drugs contain a chromophore aglycon that is derived fr om condensation of ten acetate units (catalyzed by a type II polyketide syn thase). The aglycones are glycosylated at two positions with different chai n length deoxyoligosaccharides, which are essential for the antitumor activ ity. During the early stages of mithramycin biosynthesis, tetracyclic inter mediates of the tetracycline-type occur, which must be converted at later s tages into the tricyclic glycosylated molecule, presumably through oxidativ e breakage of the fourth ring. Results: Two intermediates in the mithramycin biosynthetic pathway, 4-demet hylpremithramycinone and premithramycin B, were identified in a mutant lack ing the mithramycin glycosyltransferase and methyltransferase genes and in the same mutant complemented with the deleted genes, respectively. Premithr amycin B contains five deoxysugars moieties (like mithramycin), but contain s a tetracyclic aglycon moiety instead of a tricyclic aglycon. We hypothesi zed that transcription of mtmOIV(encoding an oygenase) was impaired in this strain, preventing oxidative breakage of the fourth ring of premithramycin B. Inactivating mtmOIV generated a mithramycin nonproducing mutant that ac cumulated premithramycin B instead of mithramycin. in vitro assays demonstr ated that MtmOIV converted premithramycin B into a tricyclic compound. Conclusions: In the late stages of mithramycin biosynthesis by Strepyomyces argillaceus, a fully glycosylated tetracyclic tetracycline-like intermedia te (premithramycin B) is converted into a tricyclic compound by the oxygena se MtmOIV. This oxygenase inserts an oxygen (Baeyer-Villiger oxidation) and opens the resulting lactone. The following decarboxylation and ketoreducti on steps lead to mithramycin. Opening of the fourth ring represents one of the last steps in mithramycin biosynthesis.