Theoretical Studies On The Mechanisms Of Several Metalloenzymes And Radical Enzymes

As a class of biological macromolecules with high catalytic activity,enzymes can efficiently catalyze various reactions under mild conditions.The research on the enzyme-catalyzed reaction helps to improve the understanding of the enzymatic reaction process,and also provides a good scientific basis for enzyme mutation,modification and design,which can expand the application of enzyme engineering in the fields of industry,agriculture and medicine.Experimenters can provide possible reaction pathways by determining crystal structure,spectral data and performing mutation studies.However,these methods are difficult to reveal the detailed mechanism of the entire catalytic reaction,as well as the related structural and energetic information of key intermediates and transition states.Through the computational methods,we can further clarify the microscopic reaction mechanism at the atomic level,and obtain the structural and energetic information of key intermediates and transition states,which can complement and verify the experimental results.With the development of experiment and computational methods,the research of enzymology has developed rapidly,especially in recent years,with the development of high-performance computers,computational method has played an increasingly important role in the study of biological macromolecules,and has become one of the important approaches in enzymology research.In this work,combined quantum mechanics and molecular mechanics method(QM/MM),molecule docking and MD simulations were employed to explore reaction details catalyzed by series of metalloenzymes and radical enzymes.Through our calculations,reaction details of these enzymes have been revealed,such as the relationship between the configuration of the initial reactants and the reaction activity,the structural and energetic information of the involved transition states and intermediates,the details of the electron transfer and the specificity of the substrate binding.In addition,we proved the important role of the nearby residues by performing mutation calculations.Based on our calculation results,we have systematically revealed the detailed catalytic mechanism of these enzymes,which provides an important theoretical basis for studying the mechanism of related enzymes,the design of inhibitors,and the study of enzyme mutations and modifications.The main research works of this dissertation are as followed:(1)A QM/MM study about the cycloclavine formation catalyzed by Aj-EasHCycloclavine is a complex ergot alkaloid containing an unusual cyclopropyl moiety,which has a wide range of biological activities and pharmaceutical applications.The biosynthesis of cycloclavine requires a series of enzymes,among which the Aj-EasH mainly responsible for the formation of the cyclopropyl group.Aj-EasH is a nonheme Fe?/?-ketoglutarate-dependent(aKG)oxidase(Aj-EasH),and the possible pathway of the formation of cyclopropyl group catclyzed by Aj-EasH has been proposed,however,the detailed mechanism of this process is still unclear.In this article,on the basis of the recently obtained crystal structure of Aj-EasH(EasH from Aspergillus japonicas),the reactant models were built,and the reaction details were investigated by performing QM-only and combined QM/MM calculations.Our calculation results reveal that the biosynthesis of cyclopropyl moiety involves a radical intermediate rather than a carbocationic or carbanionic intermediate as in the biosynthesis of terpenoid family.The iron(?)-oxo first abstracts a hydrogen atom from the substrate to trigger the reaction,and then the generated radical intermediate undergoes intramolecular ring rearrangement to form the fused 5-3 ring system of cycloclavine.On the basis of our calculations,the absolute configuration of the cycloclavine catalyzed by Aj-EasH from Aspergillus japonicus should be(5R,8R,10R),which is different from the product isolated from Ipomoea hildebrandtii(5R,8S,10S).The residues Y65,Q66,L77 and S117 near the active sites play important roles in substrate binding,ring rearrangement and enantioselectivity.(2)Theoretical insights into the inactivation mechanism of neuronal nitric oxide synthase(nNOS)The neuronal nitric oxide synthase(nNOS)is one of three isoforms of nitric oxide synthase(NOS).The other two isoforms include the inducible NOS(iNOS)and endothelial NOS(eNOS).These three isoforms of NOS are widely present in both human and other mammals,which are responsible for the biosynthesis of NO.As an essential biological molecule,NO plays an essential role in people lives,however,the overproduction of NO will cause serials of diseases,thus,the selective inhibition of three isoforms of NOS has been considered to be important in the treating of diseases.The active sites of these three enzymes are highly conserved,which is a great challenge to achieve their selective inhibition.The(S)-2-amino-5-(2-(methylthio)acetimidamido)pentanoic acid(1)has been experimentally proved to be a selective and time-dependent irreversible inhibitor of nNOS.Based on the crystal structure,experimenter has suggested the possible sulfide oxidation,oxidative dethiolation and oxidative demethylation pathways,but the details of the conversion process of this inhibitor are still unclear.In this work,we performed QM/MM calculations to verify the chemical conversion of inactivator 1.Our calculations show that the previously suggested sulfur atom as a proton acceptor to assist the C-S bond cleavage is not feasible.We designed the reaction pathway of oxidative dethiolation and oxidative demethylation mechanisms,and proposed that the imine group of the substrate could act as the proton acceptor to assist the C-S bond cleavage.These three branching reactions are competitive,but only dethiolation and demethylation reactions can generate inhibitory intermediates.As a powerful time-dependent irreversible inhibitor of nNOS,the key sulfur atom and middle imine are all necessary.Our calculation results not only verified the chemical reaction of inhibitor 1 occurred in the enzymatic active site,but also provides a reference for the design of other similar inhibitors.(3)Insight into the xylose transfer catalyzed by xyloside ?-1,3-xylosyltransferase(XXYLT1)Glycosyltransferases(GTs)are a ubiquitous group of enzymes that catalyze the synthesis of glycosidic bonds.According to the configuration changes of the anomeric carbon before and after glycosyl transfer,GTs can be divided into "retaining" and"inverting" enzymes.In this work,we performed QM/MM calculations on the retained reaction catalyzed by xyloside ?-1,3-xylosyltransferase(XXYLT1)from Mus musculus.Our calculations revealed that the previously obtained crystal structure of the UDP-Xyl ternary reaction complex is an inactive form.Accordingly,the?-phosphate oxygen O3 B of the donor should undergo a conformational change to achieve the active state,the donor and the acceptor form a strong hydrogen bond interaction to facilitatie the departure of the phosphate group.Our calculations revealed that the xylose transfer reaction follows the SNi-like mechanism,which involves a short-lived oxocarbenium-phosphate ion-pair intermediate(IP).Our calculations also revealed that two predicated transition states for the sugar-phosphate bond cleavage and glycosidic bond formation are structurally similar to the short-lived intermediate,it can be considered as a typical characteristic of the SNi-like mechanism.Q330 is responsible for stabilizing the short-lived intermediate by electrostatic interactions,and the Q330A mutant can abolish the activity of XXYLT1.In addition,using UDP-glucose as the donor,our calculations revealed that glucose transfer corresponds to a higher energy barrier due to the steric repulsion between the glucosyl moiety and the nearby residue L327.These findings not only explain the experimental observations,but also have important significance for elucidating the mechanism of GTs.(4)Anaerobic degradation of dihydroxypropanesulfonate catalyzed by DHPS-sulfolyase(HpsG)2(S)-dihydroxypropanesulfonate(DHPS)is the mainly abundant organosulfonates of the biosphere,and generated by the microbial degradation of the abundant organosulfur species 6-deoxy-6-sulfo-d-glucopyranose(sulfoquinovose,SQ).In the biosphere,photosynthesis of plants produces about 1010 tonnes SQ every year,which makes the SQ the dominant organosulfur species in nature.In addition,massive amounts of DHPS are produced by the highly abundant oceanic diatoms.With such large volumes,the degradation of DHPS has become an important role in the sulfur recycling on this planet,however,degradation of the DHPS under the anaerobic conditions is still not clear.O2-sensitive glycyl radical enzyme HpsG,distributed in both the environment and the human gut,cleavages the C-S bond and releases sulfite in the anaerobic conditions.The released sulfite will further incubate to H2S,which,however,is associated with the human health.Based on the newly obtained crystal structure of HpsG,we constructed the computational model and performed QM/MM calculations.Our calculated results revealed the degradation of the DHPS follows the radical-dependent mechanism,protein-based radical activates the substrate by extracting the hydrogen atom at the C2 position,and initiates the subsequent C-S bond cleavage.The sulfonic group is a good leaving group,and the C-S bond broken could occur directly(7.1 kcal/mol).The Glu464 is not a good proton acceptor as previous proposed,but plays an indispensable role in the proton transfer process.As the only appropriate proton acceptor of the active sites,Glu464 could relay the proton to the protein environments,which is important for the cleavage of the C-S bond.Our calculation results clarified the anaerobic degradation mechanism of HpsG,which is helpful for understanding of the anaerobic degradation of(S)-DHPS and the sulfur cycle.