Doxorubicin, a natural product isolated from Streptomyces bacteria is one of the most widely prescribed anticancer medicines. A hallmark feature of doxorubicin is cellular redox cycling of its anthraquinone moiety, which contributes both to its mechanism of action and dose-limiting cardiotoxicity. Intriguingly, the final steps of doxorubicin biosynthesis involve the P450 monooxygenase DoxA, which is dependent upon redox partners that also promote quinone redox cycling. In vitro reduction of doxorubicin by the electron donors ferredoxin and ferredoxin:NADP+ reductase leads to depletion of molecular oxygen and irreversible formation of a deglycosylated side product, 7-deoxydoxorubicinone. Recently we demonstrated that DnrV, a previously uncharacterized protein in the doxorubicin BGC, prevents this reductive activation thereby mitigating the cellular redox stress and metabolite destruction associated with redox cycling. In this study we employed multiple biochemical, biophysical, and structural techniques to elucidate the molecular basis of DnrV function. Our analyses revealed that DnrV is a multifunctional protein that simultaneously modulates the redox characteristics of the bound anthracycline and induces catalytic rate enhancement of the associated DoxA P450. These are previously uncharacterized functions of the ancient and widely conserved VOC structural fold. Through targeted genome mining, we show that VOC homologues from various non-anthracycline biosynthetic pathways replicate DnrV function, protecting both anthracycline and non-anthracycline compounds from redox cycling and activating oxidation by the DoxA P450. This work therefore characterizes DnrV, a multifunctional protein essential to doxorubicin biosynthesis and establishes DnrV as a representative member of a novel VOC functional class.

Emma Fang

University of Michigan

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