Theoretical Studies On The Catalytic Mechanism Of Several Iron-containting Metalloenzymes

Exploring the detailed mechanism of enzyme catalyzed reactions can provide important theoretical basis for enzyme engineering.The crystal structure of enzyme and the kinetic parameter of enzymatic reactions could be obtained through experimental research and the uncertain reaction pathway could be inferred,but the detailed reaction mechanism,especially the thermodynamic data of the elementary reaction could not be obtained only by experimental method,which limits the understanding of enzymatic reactions at the atomic level.Theoretical chemistry and molecular dynamics simulation have unique advantages in the analysis of reaction mechanisms,in particular,the combination of molecular mechanics and quantum mechanics has played an increasingly important role in the study of chemical reactions involving biological macromolecules,and has become an important tool for exploring the detail mechanism of enzyme catalyzed reactions.In this work,based on the crystal structure,the reaction detailed mechanism catalyzed by series of metalloenzymes were systematically explored through molecule docking,MD simulations and the combination of quantum mechanics and molecular mechanics method(QM/MM).Due to the widespread existence of iron-containing metalloenzymes in living organisms,the multi-step electron transfer and the change of iron central electron structure,it is difficult to explore the iron-containing enzymes only by experimental or theoretical researches.Based on the calculation results,some basic problems of enzymatic reactions were clarified,including the electron structure,the identification of the intermediates,the activation mechanism of substrate and oxygen,the structure and the energetics information of intermediates and the transition state.Based on the theoretical calculation results,the reaction mechanisms of some enzymes were systematically analyzed,which laid an important theoretical foundation for better understanding of the biological functions of enzymes and the modification and modification of enzymes.The main research works are as followed:(1)Insight into the Catalytic Mechanism of Nonheme Diiron Enzyme SznFSznF is a non-heme diiron-dependent enzyme with unique structure and catalytic function,which involved in the formation of N-nitrosourea moiety in the pancreatic cancer drug streptozotocin.In order to gain insights into the detailed mechanism of successive N-hydroxylation catalyzed by the central domain of SznF,the enzyme-substrate complex models were constructed through molecular docking,and the activation mechanism of oxygen by diiron was investigated.According to the calculations of the Mossbauer spectral parameters of the species,the diiron(Ⅳ)-oxo species(Q)was identified to initiate the hydroxylation reaction.Two possible pathway of hydroxylation reaction were proposed.Hydroxylation can be accomplished either by abstracting of H by Fe(Ⅳ)=O species and OH rebound or by forming the N δ-O bonds by Fe(Ⅳ)=O species attacking the N δ atom followed by intramolecular H migration.In addition,the reasons of preferential hydroxylation of Nδ-H was exploring from the theoretical view.These results may provide useful information for understanding the catalysis of SznF.(2)Insight into the Formation Mechanism of Cys-Tyr in the CDOCysteine dioxygenase(CDO)is a non-heme mononuclear iron enzyme,which catalyzes the oxidation of cysteine to cysteine sulfinic acid.Crystal structure studies of mammalian CDO showed that there is a cross-linked Cys-Tyr cofactor in the active site of the enzyme,however the formation mechanism of Cys-Tyr cross-linked structure and the influence on the catalytic efficiency are lack of thoroughly understanding.A series of computational models have been constructed,and QM/MM calculations have been performed.The calculation results reveal that the formation of Cys-Tyr in the WT-CDO contains the H-abstraction,C-S bond formation,generation of hydrogen peroxide and the aromatization of Tyr157 assisted by water molecule,and two oxygen molecules were needed in the formation of Cys-Tyr and the oxidation of cysteine catalyzed by WT-CDO.The hydrogen atoms on the benzene ring of tyrosine were replaced by F atoms to form a mutant enzyme(F2-CDO),the cofactor formation shows some differences,and one oxygen molecule was needed during the formation of Cys-Tyr and the oxidation of cysteine.In addition,the cofactor formation is just an accompanying reaction but not necessary for oxidation reaction.These results may provide useful information for understanding the catalysis of CDO.(3)Insight into the Activation Mechanism of C-H and C-F Catalyzed by LmbB2Alkaloids and its derivatives are an important nature antibiotics,and their structures usually contain five-membered cyclopyrrole.In general,the five-membered cyclopyrrole is obtained through L-tyrosine to L-3,4-dihydroxyphenylallanine(DOPA)catalyzed by tyrosine hydroxylase.When challenged with fluoro-L-tyrosine as substrate,both the C-H and C-F bonds can be hydroxylated in the same active site,but the detailed reaction mechanism has not been clarified.Based on these issues,the computational models were constructed,and a series of combined QM/MM calculations were performed.The calculation results reveal that the hydroxylation mechanisms of C-H and C-F are different.The Cpd I-like intermediate is responsible for triggering the electrophilic attack in the C-H hydroxylation.The cleavage of iron-coordinated HOOH is also in concert with the hydroxylation of C-F bond.Since the leaving F-takes two electrons away from the substrate,another molecule of hydrogen peroxide is required to complete the catalytic cycle.In addition,the calculation results indicated that it is the two binding orientations of substrate in the active site that determine the final product distribution.(4)Computational Study of Reaction Mechanism Catalyzed by UAXSUridine diphosphate(UDP)-apiose/UDP-xylose synthase(UAXS)is a special enzyme of the short-chain dehydrogenase/reductase superfamily(SDR),which catalyzes a series of complex reactions including ring opening,decarboxylation,rearrangement,ring contraction and ring closure,although UXS and ArnA which are involved in the SDR family have similar pocket of activity with UAXS,they can only catalyze the oxidation and decarboxylation reactions of UDP-GlcA.In this work,the reactant model has been constructed,and the reaction mechanism of glucuronic acid was explored.According to calculation results,the microscopic mechanism of reactions catalyzed by UAXS enzyme were explored,including that the whole reaction starts from the C4-OH oxidation,the ring-opening reaction of the substrate occurs prior to decarboxylation and the deprotonation of C2-OH group is the prerequisite for sugar ring opening.Moreover,the enol tautomerization of the decarboxylated intermediate is a necessary step for ring closure and contraction.The residues surrounding the active site assisted the catalytic reaction by participating in a series of proton transfer processes,especially the deprotonation of the C2-OH group is accomplished through a proton channel consisting of Arg182,Glu141and Asp337.The calculations may provide useful information for understanding the catalysis of the SDR family.