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(1Ⅳ)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 tctrahedral 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-mcdiated 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 prercaction-and-action mechanism in the TE macrocyclization and release of PKS product.