Theoretical Studies of Mutual Effects between 6-m-r Hemiketalization and 26-m-r Lactonization in Pimaricin Thioesterase.

Pimaricin is a small polyene macrolide antibiotic and has been broadly used as an antimycotic and antiprotozoal agent in both humans and foods. As a thioesterase in type-I polyketide synthase, pimTE controls the 26-m-r macrolide main chain release in pimaricin biosynthesis. In this work, we sought to determine whether the 6-m-r hemiketal formation was linked to pimTE-catalyzed 26-m-r lactonization. Compared to non-hemiketal TEs, pimTE is characterized by an aspartic acid residue (D179) accessible to the U-turn motif in the acyl-enzyme intermediate. Both the covalent docking and molecular dynamics simulations demonstrate that the reactive conformations for macrocyclic lactonization are drastically promoted by the 6-m-r hemiketal. Moreover, the small-model quantum mechanistic calculations suggest that protic residues can significantly accelerate the 6-m-r hemiketal cyclization. In addition, the post-hemiketal molecular dynamic simulations demonstrate that hydrogen-bonding networks surrounding the substrate U-turn of the hairpin-shaped conformation changes significantly when the 6-m-r hemiketal is formed. In particular, the R-hemiketal intermediate is not only catalyzed by the D179 residue, but also twists the hairpin structure to the 26-m-r lactonizing pre-reaction state. By contrast, the S-hemiketal formation is unlikely catalyzed by D179, which twists the hairpin in an opposite direction. Our results propose that pimTE could be a bi-functional enzyme, which can synergistically catalyze tandem 6-m-r and 26-m-r formations during the main-chain release of pimaricin biosynthesis.