A Standalone ?-Ketoreductase Acts Concomitantly with Biosynthesis of the Antimycin Scaffold

Antimycins are anticancer compounds produced by a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. The biosynthesis of these is well characterized, with the exception standalone ?-ketoreductase enzyme AntM that proposed to catalyze reduction C8 carbonyl antimycin scaffold. Inactivation antM and structural characterization suggested rather than functioning as post-PKS tailoring enzyme, acts upon terminal biosynthetic intermediate while it tethered PKS acyl carrier protein. Mutational analysis identified two amino acid residues (Tyr185 Phe223) serve checkpoints controlling substrate access active site. Aromatic checkpoint conserved in uncharacterized ?-ketoreductases, indicating they may also act concomitantly synthesis These data provide novel mechanistic insights into functionality ?-ketoreductases will enable their reprogramming for combinatorial biosynthesis.