A Cascade of Redox Reactions Generates Complexity in the Biosynthesis of the Protein Phosphatase‐2 Inhibitor Rubratoxin A

Redox modifications are key complexity‐generating steps in the biosynthesis of natural products. The unique structure of rubratoxin A ( 1 ), many of which arise through redox modifications, make it a nanomolar inhibitor of protein phosphatase 2A (PP2A). We identified the biosynthetic pathway of 1 and completely mapped the enzymatic sequence of redox reactions starting from the nonadride 5 . Six redox enzymes are involved, including four α‐ketoglutarate‐ and iron(II)‐dependent dioxygenases that hydroxylate four sp 3 carbons; one flavin‐dependent dehydrogenase that is involved in formation of the unsaturated lactone; and the ferric‐reductase‐like enzyme RbtH, which regioselectively reduces one of the maleic anhydride moieties in rubratoxin B to the γ‐hydroxybutenolide that is critical for PP2A inhibition. RbtH is proposed to perform sequential single‐electron reductions of the maleic anhydride using electrons derived from NADH and transferred through a ferredoxin and ferredoxin reductase pair.