Theoretical Study On The Mechanism Of C-C Cleavage And Hydroxylation Catalyzed By Several Iron-containing Oxidases

Enzymes can catalyze various complex metabolic reactions in life.Compared with general non-biomass catalysts,enzyme catalysis has the advantages of high efficiency,specificity and mild reaction conditions.With the development of enzymology research,enzymes are widely used in many fields such as food,fermentation,and medicine.Under the advocacy of the concept of "green chemistry",enzyme catalysis is expected to play an important role in the synthesis of renewable fuels and the biodegradation of pollutants.In-depth study of the structure and catalytic mechanism of enzymes can not only reveal the nature of biochemical reactions,but also play an inpartant role in the expanded application of enzymes and the development of enzyme engineering.Using the experimental method can obtain the crystal structure of the enzyme,the binding mode of the substrate in the active center,some reaction intermediates and the reaction kinetics,and propose the possible mechanism of the enzyme-catalyzed reaction.However,due to the complexity of enzymatic reaction,it is difficult to obtain the information of all intermediates and the transition states during the reaction only by experimental methods,and the detailed mechanism of enzyme-catalyzed reactions can not be determined.The theoretical calculations can be used to study the enzyme-catalyzed reactions at the molecular and atomic levels,and it has become an indispensable tool for the study of enzyme-catalyzed reactions.In this paper,molecular docking,molecular dynamics simulation(MD),quantum chemistry and molecular mechanics(QM/MM)methods were used to study the mechanism of carbon-carbon bond cleavage and hydroxylation reaction catalyzed by four iron-containing oxidases.We have explored the binding mode of substrate in the active center of enzyme,the role of iron center and the key residues in the catalytic reaction.In particular,the complex spin states and electron transfer of iron center during the reaction were studied.The catalytic reaction mechanisms of these iron-containing oxidases have been clarified.The results can contribute to understanding the catalytic mechanism of these enzymes,which is necessary for the related experimental research and enzyme design.The research contents of this thesis are as follows:(1).Mechanical insights into the enzymatic cleavage of double C-C bond by the latex clearing protein(LCP)The LcpK30 is a latex clearing protein(Lcp),which acts as an endo-type dioxygenase to catalyze the extracellular cleavage of the chemically inert aliphatic polymer poly(cis-1,4-isoprene),producing oligo-isoprenoids with different terminal carbonyl groups(aldehyde and ketone,-CH2-CHO and-CH2-COCH3).However,the detailed catalytic mechanism and the role of the key residues(like E148)are still unclear.In this paper,on the basis of the crystal structure of LcpK30,the enzyme-substrate reactant model has been constructed,and the cleavage mechanism of the central double bond of poly(cis-1,4-isoprene)has been elucidated by performing quantum mechanics/molecular mechanics(QM/MM)calculations.Our calculation results revealed that the oxidative cleavage reaction is triggered by the addition of the heme-bound dioxygen to the double bond of the polymer,and the pathway that involves the dioxetane intermediate was calculated to be more favorable.Three models for mutants of E148A,E148Q and E148H were constructed to explore the role of E148.Although E148 is not directly involved in the reaction,it is an important residue for fine-tuning the active pocket to accommodate the substrate for reaction.The E148 does not act as a catalytic base to extract the allylic proton to assist the reaction as previously suggested,it just contribute to construct a suitable substrate binding pocket for the addition reaction of oxygen to the substrate double bond.These findings may provide useful information for further exploring the catalysis of LcpK30,as well as the cleavage of the double bond by similar dioxygenases.(2).Mechanism of fatty acid decarboxylation catalyzed by a non-heme iron oxidase UndAUndA is a non-heme iron enzyme that was recognized to catalyze the decarboxylation of medium chain fatty acids to produce trace amounts of 1-alkenes,however,the catalytic activity is very low.According to the previous proposal,both Fe?-OO·-and Fe?=O complexes may abstract the ?-H of fatty acids to trigger the oxidative decarboxylation reaction,but the reason why its catalytic activity is very low is not clear.Herein,on the basis of the crystal structures of UndA in complex the substrate analogues,we constructed a series of computational models to explore the UndA-catalyzed decarboxylation using lauric acid as substrate.Our calculation results reveal that only the Fe?-OO·-complex can initiate the decarboxylation.In the Fe?=O complex mode,due to the relative position of the ?-H and the OFe atom is not appropriate,the H-abstraction is calculated to be difficult.For the substrate,its carboxyl group can coordinate with the Fe center in many ways,but only the monodentate coordination mode facilitates the ?-H abstraction.Due to the monodentate coordination corresponds to higher relative energy than the bidentate mode,which may explain the low efficiency of UndA.It is also reveal that as long as the ?-H is extracted by the Fe?-OO·-,the decarboxylation of substrate radical is quite easy,and an electron transfer from the substrate to the iron center is the prerequisite,which makes the decarboxylation easy to occur.These findings may provide useful information for understanding the catalysis of other non-iron decarboxylation enzymes and provide theoretical basis for the related experimental research on alkenes biosynthesis of fatty acids.(3).Theoretical study on the hydroxylation catalyzed by the nonheme diiron monooxygenase PtmU3PtmU3 is a newly identified nonheme diiron monooxygenase,which installs a hydroxyl group into the key intermediates involved in the biosynthesis of platensimycin(PTM)and platencin(PTN).The hydroxylation sets the stage for the subsequent A-ring cleavage step of diterpene derivatives.PtmU3 possesses a non-canonical diiron active site architecture with a saturated six-coordinate iron center lacking a ?-oxo bridge.Although the hydroxylation process is a simple reaction for nonheme mononuclear iron-dependent enzymes,however,how PtmU3 employs the diiron center to catalyze the H-abstraction and OH-rebound is still unknown.In particular,the electronic characteristic of the diiron is also unclear.To understand the catalytic mechanism of PtmU3,we constructed two reactant models in which both the Fe1?-Fe2?-superoxo and Fe1?-Fe2?=O are considered to trigger the H-abstraction,and performed a series of QM/MM calculations.Our calculation results reveal that PtmU3 is a special monooxygenase,i.e.,both atoms of the dioxygen molecule are incorporated into the substrate by two successive catalytic cycles.In the first cycle,Fe1?-Fe2?-superoxo catalyzes the hydroxylation reaction of the substrate,and the generated Fe1?-Fe2?=O complex can continue to catalyze the hydroxylation reaction of the substrate in the second round.In the diiron center,Fel only plays a structural role and Fe2 adopts the high spin state(S=5/2)during the catalysis.E241 and D308 not only act as bridging ligands to connect two Fe ions but also take part in the electron reorganization.Obvious electron transfer was observed in the OH-rebound reaction,which can explain the lower barrier of hydroxylation.This study can illuminate the hydroxylation mechanism catalyzed by PtmU3,and provides some important information for the study of the catalytic reactions of other non-heme diiron enzymes.(4).Theoretical study on the hydroxylation of 4-Cl-o-cresol catalyzed by the hemoglobin dehaloperoxidase(DHP B).The hemoglobin dehaloperoxidase(DHP B)from the Amphitrite ornata can catalyze the dechlorination and hydroxylation of 4-Cl-o-cresol,which is the basis for the degradation of 4-Cl-o-cresol.On the basis of the crystal structure,the catalytic reaction details of DHP B have been elucidated.Accoding to the calculated results,the favaroble pathway was obtained,in which His55 abstrated the the proton on the hydroxyl group of the substrate to initiate the reaction,then the proton is transferred to the Fe=O center,and finally the OH group rebound to the substrate.In the catalytic reaction,Fe=O center not only completes H-abstraction,but also acts as the electron acceptor.These findings will provide useful information for further understanding of the catalytic action of DHP B and for exploring the reaction mechanism of biodegradable pollutants.