Insight Into Substrate Selectivity And Catalytic Mechanism Of Pimaricin Thioesterase

As the last domain of polyketide synthase（PKS）,thioesterase（TE）regulates macrocylcization/hydrolysis and release of product.It was reported that with Pim G knocked out from pimaricin PKS,three derivatives were produced with higher efficacy and lower toxicity,yet unsatisfactory yield.Here,MD simulations and QM/MM calculations were combined to reveal the interaction between pimaricin TE（pima-TE）and its natural and unnatural substrates.Previous work on type I PKS has indicated a“pre-reaction state”（PRS）of protein-substrate complex,which was decisive to the proceeding of cyclization,could form prior to reaction.In the dissertation,the conformational transition,recognition pattern and product release between pima-TE and C₁₂methyl-substituted substrate were studied,and key residues involved in mutual recognition were described.Next,pima-TE’s catalytic disparity between natural and unnatural substrates were analyzed.Both ones had similar proportion of PRS formation,while they differed in intermediate conformation and catalytic mechanism.Last,an experimental phenomenon that L170R mutation on pima-TE would divert the reaction from macrocyclization to hydrolysis has also aroused our curiosity.A detailed mechanism accounting for pima-TE macrocyclization/hydrolysis selectivity was provided via MD simulations,and QM/MM calculations testified the feasibility of hydrolysis in L170R mutant.Not only did our research advance the development of new antigfungal drugs,but we also laid a theoretical foundation for the clarification of TE catalysis from other polyene polyketides.