Theoretical Insights Into Catalytic Mechanisms Of Several Iron-containing Enzymes

Enzymes are biological macromolecules with catalytic function produced by living cells,which are called as biocatalyst.Compared with the general catalysts,the enzymes have many advantages:high catalytic activity,high specificity and activity regulated by some compounds.The metal ions are usually acted as the coenzymes in the active sites,and the metal ions contain magnesium ions,zinc ions,iron ions,manganese ions,cobalt ions,calcium ions,etc.The Fe-containg enzymes are the common metal enzymes,including dioxygenase(Catechol oxidase),monooxygenase(Cytochrome P450 enzyme,Methane monooxygenase),peroxidase(Horseradish peroxidase),catalase(Catalase),dismutase(Fe-Superoxide dismutase).Because the Fe-containg enzymes catalytic reactions are very fast,the reaction mechanism details are difficult to be captured by experimental means.With the development of the theoretical chemistry methods,we can conduct the study of reaction mechanism of the enzyme from the atomic level.In this dissertation,the combinded quantum mechanics/molecular mechanics(QM/MM)method is to explore the mechanism of several Fe-containing enzymes.According to the QM/MM results,the catalytic mechanism is first determined,and the stable structures along the reaction coordinate with the kinetic information are obtained.Furthermore,we explored the important role of some residues in the active site.Calculation results are in good agreement with the experimental results,which may provide some guidance for the further study of related enzymes family.The main research works of this dissertation as followed:(1)Insights into the catalytic mechanism of tryptophan lyase(NOSL).L-Tryptophan lyase(NosL),a member of the radical S-adenosyl-L-methionine(SAM)-dependent superfamily,catalyzes the conversion of L-tryptophan to 3-methylindolic acid(MIA).In this article,on the basis of the recently obtained crystal structure of NosL(PDB code 4R34)in 2014,a combined quantum mechanical/molecular mechanical(QM/MM)approach has been employed to elucidate the reaction details,involving substrate amine dehydrogenation,C-C bond cleavage and carboxyl fragment migration.Our results show that the hydrogen in the amino group of L-tryptophan is suitable for abstraction by the Ado radical,and this step corresponds to an energy barrier of 12.4 kcal/mol.Two possible modes of C-C cleavage(pathl and path2)have been considered.The cleavage of the Ca-C? bond is thermodynamically more favorable than the cleavage of the Ca-C bond with their energy barriers being 7.2 and 15.0 kcal/mol,respectively.And the easy breaking of Ca-C? may be attributed to the electron hole delocalization in the amine radical of the substrate.The intermediate derived from the cleavage of the Ca-C bond is calculated to be a stable species,and the cleavage of the Ca-C bond is accompanied by the migration of the 'COO-fragment.These conclusions are basically in accordance with EPR-trapped analysis and can account for the absence of the 'COO-fragment.The shunt product(3-methylindole)is obtained from the cleavage of the Ca-C? bond with an energy barrier of 19.5 kcal/mol.However,the rate limiting step is the formation of 3-methylindolic acid(MIA),which corresponds to an energy barrier of 26.9 kcal/mol.Our investigations thus give a better comprehension of the NosL reaction mechanism and may contribute to the understanding of the SAM superfamily.(2)The epoxidation mechanism of fumitremogin B endoperoxidase(FtmOxl).Fumitremorgin B endoperoxidase(FtmOxl)from Aspergillus fumigatus is the first reported a-ketoglutarate dependent mononuclear non-haem iron enzyme that catalyzes the endoperoxide formation reaction,converting Fumitremorgin B to verruculogen.Experiments reveal that the molecular oxygen(02)is incorporated into verruculogen without O-O bond scission,which differs from the currently known non-haem iron enzymes,but the mechanistic details are still unclear.In this paper,on the basis of the crystal structures of FtmOxl in complex with either the co-substrate(a-ketoglutarate)or the substrate(fumitremorgin B),a ternary complex model of the enzyme-a-ketoglutarate-substrate has been constructed,and combined quantum mechanics and molecular mechanics(QM/MM)calculations have been performed to unravel the novel mechanism of FtmOxl.Our calculations indicate the quintet of the FeIV=O complex as the ground state.The FeIV=O complex firstly abstracts a hydrogen from the hydroxyl of Tyr228 to initiate the reaction,which corresponds to a lower energy barrier(9.1 kcal/mol).If the FeIV=O complex directly abstracts a hydrogen from C21 of the substrate,the energy barrier would increase to 33.9 kcal/mol.When Tyr228 was mutated to Ala228,this energy barrier decreases to 24.0 kcal mol-1.In the subsequent reaction,the generated Tyr228 radical abstracts the hydrogen(H2)from C21 of the substrate with an energy barrier of 23.8 kcal/mol.The second molecular oxygen binds to the C21 radical of the substrate in the active pocket and further completes the epoxidation with an energy barrier of 4.8 kcal/mol.These results may provide useful information for understanding the reaction mechanism of FtmOxl and provide guidance for further experimental investigations.(3)The oxidative rearrangement mechanism of P450 CYP162C2(PntM).The CYP162C2(PntM)from Streptomyces arenae is a member of cytochrome P450 enzymes,which catalyzes the unusual oxidative rearrangement of pentalenolactone F(1)to the sesquiterpenoid antibiotic pentalenolactone(3).Based on the crystal structure of PntM bound with substrate(pdb code:5L1O),the quantum mechanical/molecular mechanics(QM/MM)calculations have been performed to explore the detailed mechanism of PntM-catalyzed oxidative rearrangement.The conversion from pentalenolactone F(1)to pentalenolactone(3)involves the stereospecific removal of the H-1si from 1,the syn-1,2-migration of the 2si methyl group,and the antarafacial loss of H-3re.The abstraction of H-lsi by Cpd I is calculated to be rate limiting with an energy barrier of 20.3 kcal/mol,which agrees with estimated free energy barrier from the experiments(18.6 kcal/mol).It is the unfavorable geometry of Fe-OH-C1 that blocks the oxygen rebound reaction,and the following intramolecular syn-1,2-methyl migration is accompanied by an electron transfer from the substrate to the porphyrin ring via Fe-OH group,generating the carbocation intermediate.Owing to the positive charge,the intermediate can easily loss a proton to form the final products.Our calculation results indicate that both the carboxyl group of porphyrin and Fe-OH can act as the base to accept the proton of the substrate,and the previous proposal that the arginine Arg74 acts as the Bronsted base is unlikely.The target product pentalenolactone and the three isomeric by-products correspond to four different modes of deprotonation.(4)The mechanism of decarboxylative hydroxylation of salicylate catalyzed by the Flavin dependent monooxygenase salicylate hydroxylase.Salicylate hydroxylase(SALH)is a Flavin-dependent monooxygenase responsible for the transformation of salicylate to catechol.In this article,on the basis of the crystal structure(PDB code:5evy)obtained from Pseudomonas putida S-1,combined quantum mechanical/molecular mechanical(QM/MM)calculations have been performed to investigate the reaction mechanism of SALH.Since the formation of C4a-hydroperoxyflavin has been theoretically confirmed to be a barrierless process,our calculations started from the C4a-hydroperoxyflavin intermediate.The whole enzymatic reaction can be divided into two parts:the hydroxylation and decarboxylation.Our calculation results indicate that the deprotonated substrate is the active form,whereas the neutral form of salicylate corresponds to very a high energy barrier(39.8 kcal/mol)for the hydroxylation process.The deprotonated salicylate tends to be present in weakly alkaline mediumwhich is in line with the experimental result that the optimum pH is 7.6.The calculated results with the deprotonated substrate indicate that the hydroxylation and decarboxylation occur in a stepwise manner,and the decarboxylation process is calculated to be the rate-limiting step with an energy barrier of 14.5 kal/mol.Calculations using different functionals(B3LYP,BP,BVWN,PBE,M06 and TPSSH)suggest the catalytic reaction is highly exothermic,which is consistent with its similar enzyme(PHBH).