Theoretical Studies On Catalytic Mechanism Of Dehydratase/Thioesterase In Type ? PKSs

Polyketide synthetases(PKSs)deploy a modular biosynthetic strategy to produce diverse molecules that have important medicinal utility such as antibiotics,anti-cancer agents,and immunosuppressants,et al.Combined with computational biology,directed evolution has accelerated the research process of enzyme modification,such as modification of substrate specificity and thermal stability of enzymes.In this study,the structural domains of dehydrase(DH)and thioesterase(TE)in type ? PKS were studied by MD simulation and QM/MM methods,thus providing theoretical guidance for its rational design and directed evolution.DH,a domain in polyketide synthase modules,can catalyze the dehydration of ?-hydroxy to a ?,?-unsaturated acyl intermediate.As the first dual-function(dehydratase/isomerase)DH domain accessible in PDB database,gephyronic acid(Gph F)DH1 domain from the PKS biosynthetic pathway attracts great concerns of researchers.However,the mechanisms of dehydration and isomerization in type ? PKS remain unclear.In this study,MD simulations and QM/MM calculations were combined to elucidate the molecular mechanism of Gph F DH1.The results indicated that Gph F DH1 has better recognition effect towards(2R,3R)-substrate and prefers catalyzing the ?-? double bond firstly to forming ?-? double bond directly.By calculating the binding energy,13 key residues near the active pocket were highlighted.Umbrella sampling results indicated that nonmethylated substrate forms intra-molecular hydrogen bond more easily than ?-methyl substrate.The QM/MM calculations with M062X/6-311+G**//M062X/6-31G* method and SMD solvation correction supported one-base dehydration and one-base isomerization mechanism with energy barriers of25.3 kcal/mol and 17.2 kcal/mol,respectively.These results encourage future studies for the full comprehension of catalytic mechanism of PKS DHs and for the rational design of typical DHs.Tautomycetin(TMC)is a naturally linear polyketide metabolite with a variety of pharmacological activity.An experiment,swapping of TMC thioesterase domain with pikromycin(PIK)TE,indicates that not only linear TMC precursor,but also cyclized TMC was produced.In this study,we attempted to uncover the reasons why TMC TE prefers hydrolysis rather than macrocyclization,and reveal the molecular basis of TE-catalyzed hydrolysis and macrocyclization.Pre-reaction states(PRSs),which are critical to macrocyclization,were observed in both systems.Residue Y161 in TMC TE was noticed by analyzing hydrogen bond and hydrophobic interactions,which seemed to inhibit the effective deprotonation of the substrate,and block the nucleophilic attack owing to steric hindrance.The additional simulations indicated that Y161 A mutant might improve the cyclization by increasing the proportion of PRS.Besides,more water molecules appeared around the active site of TMC-TE system,which might improve the probability of hydrolysis.Additionally,the QM/MM calculations indicated that compared to macrocyclization,hydrolysis readily takes place.The barrier of hydrolysis was calculated to be 13.5 and 11.0kcal/mol in TMC-TE and PIK-TE systems,respectively.Besides,macrocyclization may hardly occur in TMC TE(39.6 kcal/mol),while it may slightly proceed in PIK TE(24.4 kcal/mol).Our study elucidates the molecular basis of the catalytic mechanism of TMC TE and PIK TE and furthers the discovery and design of novel engineered macrolactones.