Angucyclines are a structurally diverse class of actinobacterial natural
products defined by their varied polycyclic ring systems, which display
a wide range of biological activities. We recently discovered
lugdunomycin (1), a highly rearranged polyketide antibiotic
derived from the angucycline backbone that is synthesized via several
yet unexplained enzymatic reactions. Here, we show via in vivo, in vitro,
and structural analysis that the promiscuous reductase LugOII catalyzes
both a C6 and an unprecedented C1 ketoreduction. This then sets the
stage for the subsequent C-ring cleavage that is key to the rearranged
scaffolds of 1. The 1.1 Å structures of LugOII in complex with either ligand 8-O-Methylrabelomycin (4) or 8-O-Methyltetrangomycin (5)
and of apoenzyme were resolved, which revealed a canonical Rossman fold
and a remarkable conformational change during substrate capture and
release. Mutational analysis uncovered key residues for substrate
access, position, and catalysis as well as specific determinants that
control its dual functionality. The insights obtained in this work hold
promise for the discovery and engineering of other promiscuous
reductases that may be harnessed for the generation of novel
biocatalysts for chemoenzymatic applications.