The actinomycins constitute an interesting class of natural products in which the yellow-red phenoxazinone chromophore is linked to two cyclic pentapeptides. These compounds are among the most potent antitumor agents known. Their clinical use however, has been limited by their high toxicity, to the treatment of choriocaracinoma, Wilms tumors, rhabdomysoarcoma and Kaposi's sarcoma. The mechanism of their cyto-toxicity has been intensively studied. Actimomcyin binds to DNA by intercalation of the phenoxazinone between adjacent GC base pairs. This interaction results in highly specific inhibition of DNA dependent RNA synthesis. Because of this specificity actinomycins have also been widely used as biochemical tools. In this proposal we will focus on the mechanism of phenoxazinone synthase. This enzyme catalyses the oxidative coupling of two molecules of aminophenol to form the phenoxazinone chromophore and is responsible for the last step in actinomycin biosynthesis. This reaction is a complex oxidative coupling for which no mechanistic information is available. Our strategy involves a combination of biophysical techniques, kinetic studies with mechanistically probing altered substrates, active site sequence determination and oligonucleotide directed point mutagenesis of the active site.
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