Direct Evidence That 8-Dehydrogenation Channels the Biosynthetic Pathway of Rifamycins

The 8-OH of rifamycin is essential for its bioactivity, while its naphthalene ring formation with 8-OH has remained unclear. Biochemical and structural analysis has demonstrated that a pair of rare NAD⁺-dependent dehydrogenases, RifS and RifT, forms a S₂T₂ structure to dehydrogenate at C8 in a structure-dependent manner. RifS catalyzes 8-dehydrogenation through a canonical NAD⁺-dependent mechanism, while RifT acts as a noncatalytic partner. Finally, we proposed a plausible pathway for the transformation of benzene-type prorifamycin A (1) to naphthalene-type 34a-deoxyrifamycin W (1a) bearing the 8-OH group. These results provided direct evidence for the branch point of rifamycin and 8-deoxyrifamycin biosynthesis and paved an approach to engineering novel ansamycins.