An Evolutionarily Conserved Protein Fold with Multiple Roles in Anthracycline Biosynthesis

Doxorubicin, a glycosylated type II PKS-derived natural product first isolated from Streptomyces peucetius is a widely prescribed anticancer medicine and cytotoxic agent. It is also the final oxidation product of DoxA, a cytochrome P450 that catalyzes three sequential C–H oxidation reactions at the C13 and C14 positions on deoxy-daunorubicin leading to doxorubicin. A key limitation of in vitro P450 biocatalysis with anthracycline substrates is the rapid reductive de-glycosylation in the presence of electron donors like NADPH and ferredoxin reductase leading to inactive aglycones. This undesired reaction derails late-stage oxidative functionalization of anthracycline derivatives for clinical development. Recently, we demonstrated that DnrV, a previously uncharacterized vicinal oxygen chelate (VOC) protein in the doxorubicin BGC, prevents reductive activation thereby mitigating the cellular redox stress and metabolite destruction associated with redox cycling. In this study, we employed biochemical, biophysical, and structural methods 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 iterative cytochrome P450 DoxA. These are novel functions of the ancient VOC protein family, representing a new functional class we term REdox-Associated CytoToxin binding protein (REACT). Homologues of DnrV from non-anthracycline BGCs display similar redox-protective activity, and some extend this function to the bacterial metabolite menadione. These findings establish REACTs as a conserved family of multifunctional proteins that modulate quinone redox behavior and promote oxidative tailoring of redox-active natural products.