Mechanistic insights into double-bond migration mediated by bifunctional dehydratases in ansamitocin biosynthesis

Abstract

Ansamitocin, a potent antitumor polyketide compound from Actinosynnema pretiosum, is assembled by a modular type I polyketide synthase (PKS-I). During its biosynthesis, a unique double-bond migration coupled with the canonical β-hydroxy dehydration is identified and proposed to be catalyzed by two dehydratase domains (DH2 and DH3). This rearrangement is widespread during the biosynthetic pathways of numerous natural polyketides, endowing them with structural diversity and enhanced bioactivity. Here, we employed molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations to elucidate the detailed mechanisms of double-bond formation and migration catalyzed by dehydratases DH2 and DH3 in ansamitocin biosynthesis through dehydration and isomerization. In addition to the catalytic residue, key interactions between DHs and their substrates in dominant conformations obtained from MD simulations were identified. The energy barriers for the dehydration and isomerization reactions catalyzed by DH2 were calculated to be 16.0 and 19.5 kcal mol⁻¹, respectively, whereas those for the same two steps catalyzed by DH3 were 16.1 and 19.3 kcal mol⁻¹. The isomerization constitutes the rate-determining step in both cases. In contrast to previously proposed one-base mechanisms, our findings support a base–acid cooperative mechanism operative in both dehydration and isomerization. These atomic-level insights reveal how adjacent DH domains coordinate sequential double-bond formation and migration—a widespread biosynthetic strategy that enhances the biological activity of polyketides.