Theoretical Study On The Catalytic Mechanism Of Thioesterase-catalyzed Cyclization And Hydrolysis In Polyketide Synthase

Polyketide synthases(PKS)and non-ribosomal polypeptide synthetases(NPRS)synthesize many important pharmaceutical molecules.The synthases contain multiple catalytic cores,including a starter module,several extension modules,and termination module.In general,thioesterases act as a termination module responsible for the product release.Thioesterase can catalyze both the cyclization(lactonization)and hydrolysis of the substrates,where the cyclization(lactonization)products are the biosynthetic target molecules and hydrolysis products are by-products.So the ratio of the two products is determined by the chain length,the chirality of substrates.The goal of this dissertation is to improve the biosynthetic efficiency of the target macrocyclization products.Through molecular simulation and data analysis,the cyclization and hydrolysis mechanism of thioesterases were studied.The dissertation explains the cyclization mechanism of substrate 30(N-acetylcysteamineseco-10-deoxymethynolide)catalyzed by DEBS TE and proposes the concept of “prereaction state” to elucidate the chemical selectivity.The substrate 30 can reach the prereaction state by the interaction with the “open-close” motion of thioesterase pocket and the conformational change needs to climb 11.6 kcal/mol energy barrier of structural rearrangement.The prereaction structure can stay stably for a certain period of time in the molecular simulation,which gives the terminal hydroxyl group a chance to attack the C1 carbonyl carbon and makes the substrate form a reactive tetrahedron intermediate with a 9.9 kcal/mol energy barrier.The unstable tetrahedral intermediate traverses a small energy barrier and releases a cyclic product eventually.While the only difference between the substrate 31/32(N-acetylcysteamine thioester of seco-7-dihydro-10-deoxymethynolide)and the substrate 30 is that the carbonyl group at position 7 on the 10-deoxymethynolide backbone in substrate30 is replaced with a hydroxyl group in substrate 31/32,the substrate 31/32 failed to form a prereaction structure.More importantly,the substrate tail of 31/32 prefers to move to the pocket exit which leads to the accessible of water,so hydrolytic reaction is more reachable.Based on the previous studies of DEBS TE,we further accomplish a contrastive research in PICS TE which is the homologous protein of DEBS TE.Although the prereaction conformation also appears in the complex between PICS TE and substrate 30,the occurrence frequency of prereaction state is less than that in DEBS TE.In PICS TE,the hydroxyl group in the substrate tail form a hydrogen bond with the carbonyl oxygen atom at the C1 position of the substrate 30,in contrast,the hydrogen bonding in prereaction state is between the hydroxyl of the substrate tail and histidine in the catalytic triplet.The hydrogen bonding in PICS TE belongs to an intramolecular hydrogen bonding and thus the conformation is named as preorganization state.The non-reactive preorganization state decreases the occurrence frequency of prereaction state.From the appearance time and energy changes of the two key conformations,it can be inferred that the preorganization state can rapidly transform to the prereaction state by climbing a relatively small energy barrier,and ultimately promotes the cyclization of the substrate 30.The cyclization reaction following the prereaction state shows a slight difference in energy from that in DEBS TE.The energy barrier for forming TS1 is 11.1 kcal/mol which is higher than that in DEBS TE.The tetrahedral intermediate can release the final cyclization product by overcrowding a 2 kcal/mol energy barrier.The dissertation also contributes the effect to elucidating the substrate stereoselectivity in C11 hydroxyl when catalyzed by DEBS TE.The substrate 10(S-2-Acetamidoethyl 7-((3R,5R)-3,5-dihydroxy-6-phenylhexanamido)heptanethioate)is similar to the natural substrate of DEBS TE(substrate 1)and can also form a prereaction conformation by interaction with the DEBS TE.However,the prereaction state is less stable,and the terminal hydroxyl often escapes the hydrogen bonding which constituted with the catalytic histidine.As a result,the occurrence frequency of prereaction state is strongly decreased.By QM/MM calculation,the escaping from prereaction of substrate 10 is an energy decreasing process,which explains the instability prereaction state of the substrate 10.In addition,with the participation of water molecules,the escaping rate of substrate 10 become faster and the situation aggravates the tendency of hydrolysis side reaction.The catalysis property of substrate11(S-2-Acetamidoethyl 7-((3S,5R)-3,5-dihydroxy-6-phenylhexanamido)heptanethioate)is consistent with the substrate 30/31 in DEBS TE.It is a common phenomenon in these substrates that the tail of these substrates often move to the pocket exit in cooperation of DEBS TE,thus failing to form a prereaction state.The binding models between different substrates and different enzymes in the prereaction state were further summarized.Two important hydrophobic amino acids: Ala77(Ala78)and Phe260(Phe269)are conservated in DEBS TE and PICS TE.Ala77(Ala78)is in the center of the pocket,and helps the substrates turn its tail back in the middle;Phe260(Phe269)is located beside the catalytic histidine.On the one side,Phe260(Phe269)help the hydroxyl of the substrate obtain a correct orientation and form hydrogen bonding with catalytic histidine.On another side,Phe260(Phe269)form a hydrophobic gate with the substrate tail and prevent the water molecules from the catalytic center.The distribution of hydrophobic amino acids which interacts with the substrate in DEBS TE was dispersed and mainly distributed in the L1 region and the loop region between the ?3 fold and ?1 helix.However,the hydrophobic amino acids of PICS TE are mainly distributed in the two ?-helical regions of the Lid region,that is,the L1 and L2 regions.Thus,although PICS TE is very similar to DEBS TE,its hydrophobic core is using a different strategy.This dissertation demonstrates the dynamic induce-fit process between the thioesterases and the substrates,proposes the “pre-reaction” which is the key structure before the cyclization reaction,and points out the key hydrophobic amino acids alongside the reaction.These findings will provide new ideas for the thioesterase design and optimize the catalytic efficiency of thioesterase.