Theoretical Studies on the Catalytic Mechanism and Substrate Diversity for Macrocyclization of Pikromycin Thioesterase

Abstract

Polyketide synthases (PKSs) share a subset of biosynthetic steps in construction of a polyketide, and the offload from the PKS main module of specific product release is most often catalyzed by a thioesterase (TE). In spite of the fact that various PKS systems have been discovered in polyketide biosynthesis, the molecular basis of TE-catalyzed macrocyclization remains challenging. In this study, MD simulations and QM/MM methods were combined to investigate the catalytic mechanism and substrate diversity of pikromycin (PIK) TE with two systems (PIK-TE-1 and PIK-TE-2), where substrates 1 and 2 correspond to TE-catalyzed precursors of 10-deoxymethynolide and narbonolide, respectively. The results showed that, in comparison with PIK-TE-2, system PIK-TE-1 exhibited a greater tendency to form a stable prereaction state, which is critical to macrocyclization. In addition, the structural characteristics of prereaction states were uncovered through analyses of hydrogen-bonding and hydrophobic interactions, which were found to play a key role in substrate recognition and product release. Furthermore, potential energy surfaces were calculated to study the molecular mechanism of macrocyclization, including the formation of tetrahedral intermediates from re- and si-face nucleophilic attacks and the release of products. The energy barrier of macrocyclization from re-face attack was calculated to be 16.3 kcal/mol in PIK-TE-1, 3.6 kcal/mol lower than that from si-face attack and 4.1 kcal/mol lower than that from re-face attack in PIK-TE-2. These results are in agreement with experimental observations that the yield of 10-deoxymethynolide is superior to that of narbonolide in PIK TE catalyzed macrocyclization. Our findings elucidate the catalytic mechanism of PIK TE and provide a better understanding of type I PKS TEs in protein engineering.

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

“…In short, the nucleophilic attack to the formation of tetrahedral intermediate was determined to be the ratedetermining step, and the energy barrier was also in good agreement with that reported energy barrier of picromycin TE-catalyzed macrocyclization [25]. These results indicated that the macrocyclization catalyzed by NYS TE with its natural substrate Nys could favorably take place in both thermodynamic and kinetic aspects.…”

“…The transition state TS1 was located at the d 1 =1. To sum up, the whole energy barrier of the cyclization was 14.0 kcal/mol, which is similar to the barrier (16.3 kcal/mol) of 10-deoxymethynolide macrocyclization catalyzed by picromycin TE [25]. These results indicated that AMB-TE can easily catalyze this macrocyclization with its natural substrate Amb.…”

“…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].…”

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.…”

Insight into Structural Characteristics of Protein-Substrate Interaction in Pimaricin Thioesterase

“…Since the transition states of enzyme catalysis were hard to obtain in silico, we chose pre-reaction state (PRS) as an evaluation indicator. According to our previous research [ 26 , 36 ], PRS was the very prior stage of macrocyclization in terms of both structure and energy, and its formation was decisive to TE cyclization. The proportion of PRS was regarded as the degree of reaction readiness.…”