Theoretical Studies on the Mechanism of Thioesterase-Catalyzed Macrocyclization in Erythromycin Biosynthesis

Abstract

Macrocyclic polyketides, biosynthesized by modular polyketide synthases (PKSs), have been developed successfully into generation-by-generation pharmaceuticals for numerous therapeutic areas. A great effort has been made experimentally and theoretically to elucidate the biosynthesis mechanisms, in particular for thioesterase (TE)-mediated macrocyclization, which controls the final step in the PKS biosynthesis and determines chemical structures of the final products. To obtain a better insight into the macrocyclization process (i.e., releasing step), we carried out MD simulations, QM and QM/MM calculations on complexes of 6-deoxyerythronolide B synthase (DEBS) TE and two substrates, one toward a macrocyclic product and another toward a linearly hydrolytic product. Our investigation showed the induced-fit mutual recognition between the TE enzyme and substrates: in the case of macrocyclization, a critical hydrogen-bonding network is formed between the enzyme and substrate 1, and a hydrophobic pocket appropriately accommodates the substrate in the lid region, in which a pivotal prereaction state (1 IV′) with an energy barrier of 11.6 kcal/mol was captured on the potential energy surface calculation. Accompanied with the deprotonation of the prereaction state, the nucleophilic attack occurs with a calculated barrier of 9.9 kcal/mol and leads to the charged tetrahedral intermediate. Following the decomposition of the intermediate, the final macrocyclic product releases with a relatively low barrier. However, in the case of hydrolysis, such a prereaction state for cyclization was not observed in similar molecular simulations. These calculations are consistent with the previous biochemical and structural studies about the TE-mediated reactions. Our study indicated that the enzyme–substrate specificity stems from mutual molecular recognition via a prereaction state between DEBS TE and substrates, suggesting a prereaction-and-action mechanism in the TE macrocyclization and release of PKS product.

Understanding the R882H mutation effects of DNA methyltransferase DNMT3A: a combination of molecular dynamics simulations and QM/MM calculations

“…32 In our recent papers, the pre-reaction states (PRSs), complexes of the substrate and the active site reactant, were successfully used to understand the substrate diversity of thioesterase. 33,34 Here, the pre-reaction state (PRS) is also employed to understand the effects of R882H mutation on DNMT3A methylation. In practice, the pre-reaction state is dened as the product of the nucleophilic attack of Cys710 on cytosine.…”

Insights into Specificity and Catalytic Mechanism of Amphotericin B/Nystatin Thioesterase

“…3.0Å and d (C 1 -O sub ) [?] 4.5A were defined as Pre-Reaction States (PRSs) in our previous works [25,26]. The PRS has been wildly used in structural comparisons and mutant analyses [39][40][41].…”

“…Our previous studies [26] have shown that the thioesterase-substrate system will reach Pre-Reaction States (PRSs) before the cyclization reaction occurs. In this special state, histidine and aspartic acid in the catalytic triad constitute a proton transfer chain to facilitate the deprotonation of the distal hydroxyl group of the substrate and further promote the intramolecular nucleophilic attack to obtain a tetrahedral intermediate.…”

Towards Precision Engineering of Canonical Polyketide Synthase Domains: Recent Advances and Future Prospects

“…In the absence of clear crystal structures, detailed theoretical approaches have been undertaken to model both cyclization and hydrolysis reactions catalyzed by DEBS TE. Chen et al used QM and MM models to investigate determinants of the reaction fate of three DEBS-like SNAC substrates [ 96 ]. Their work suggests that the reaction is mediated through an induced-fit mutual recognition between the TE enzyme and substrates.…”