Theoretical Studies On The Catalytic Mechanisms Of Several Non-heme Oxidases

In the body,various kinds of reactions involved in the metabolism are catalyzed by enzymes.Compared with the traditional catalysts,enzymes have many significant advantages,such as high efficiency,high specificity of catalytic reactions and mild reaction conditions.With the continuous development of enzymology,enzymes have been widely used in medicine,food,light industry,environment protection and some related scientific research fields.Furthermore,chemical means,structural biology,bioinformatics and genetic manipulation technologies have developed rapidly in recent years.Researchers can make enzyme more suitable for the pharmaceutical,industrial and agricultural demand by modifying,transforming,designing and developing the enzyme molecules.Therefore,it is necessary to investigate the structure,properties,function and reaction mechanism of enzymes,which will be helpful to clarify the nature of the life,understand the biological function of the enzymes and expand the application field of the enzymes.In the enzymatic reaction researches,experimental methods can be used to obtain some important data,such as the rate values of catalytic reaction,the crystal structures,the kinetic parameters,the mutation results and so on.Theoretical calculation results can provide the structures of the transition states and intermediates,as well as the energetic information involved in the enzymatic reaction,which are difficult to be captured experimentally.Thus the theoretical calculations can be used to supplement and explain the experimental results.With the continuous progress of the computer technology and the algorithm development,the computational chemistry has become an important tool in the investigation of large-scale biochemical systems.In this work,the molecule docking,molecular dynamics simulation and the combination of quantum mechanics/molecular mechanics method(QM/MM)were performed to explore several kinds of non-heme oxidases.All of the oxidases in this work contain the metal co-factor.Because the metalloenzymes are widely distributed and the electronic structures of metal ions are more complicated,the theoretical research about the metalloenzyme is attractive and difficult.This work is focused on solving some key questions involved in the enzymatic reaction,such as determine the possible binding modes of the substrates in the active sites,obtain the structures of the transition states and intermediates,clarify the optimal reaction pathway and analyze the effect of some key residues and ligands.This work can illuminate the catalytic mechanism at the atomistic level,which may contribute to understanding the biological function and the application of enzymes.The main contents in this work are as follow.(1)Mechanistic investigation of isonitrile formation catalyzed by ScoERecent structural and biochemical evidence showed that ScoE from S.coeruleorubidus is a non-heme iron/a-KG dependent decarboxylase,which catalyzes the formation of isonitrile group by desaturation and decarboxylation.This discovery offers an alternative mechanism for isonitrile formation.The other isonitrile synthases,such as IsnA,XnPvcA or AmbI1/AmbI2,convert R-CH(-NH2)-CO2-to R-CH(-N--C)-CO2-by introducing additional carbon unit,however,ScoE catalyzes the conversion of R-NH-CH2-CO2-to R-N-?C through oxidative decarboxylation.To explore the catalytic mechanism of ScoE,on the basis of the high-resolution crystal structure,the enzyme-substrate complex models were constructed,and a series of combined QM/MM calculations were performed.Our results reveal that the ScoE-catalyzed reaction contains two decoupled desaturation and decarboxylation The Fe?-oxo-triggered desaturation includes two consecutive H-abstractions,which are similar to the C-C single bond desaturation catalyzed by other non-heme iron/a-KG dependent desaturases.In the second stage reaction,the decarboxylation of substrate radical generated by H-abstraction was calculated to be quite easy,whereas the previously proposed decarboxylation that involves the hydroxylated intermediate was calculated to be difficult.Importantly,the electron transfer from the substrate to the iron center is the key factor for lowering the barrier of decarboxylation.Thus,the central iron ion is not only responsible for H-abstraction,but also acts as an electron relay station for decarboxylation.In addition,this electron transfer was found to be coupled with a proton transfer,in which R310 and the associated H-bonding network play a critical role.In general,the first C-N desaturation is the rate-limiting step of the whole catalysis with an overall energy barrier of 17.6 or 16.9 kcal/mol in two competitive pathways,qualitatively agreeing with the estimated free energy(17.9-18.1 kcal/mol)from experiments.These results may provide useful information for understanding the biosynthesis of isonitrile and the oxidative decarboxylation catalyzed by non-heme iron/a-KG dependent enzymes.(2)Mechanistic study of two nogalamycin synthases SnoK and SnoNThe non-heme iron/a-ketoglutarate dependent enzymes SnoK and SnoN from Streptomyces nogalater are involved in the biosynthesis of anthracycline nogalamycin.Allhough they have similar active sites,SnoK is responsible for carbocyclization whereas SnoN solely catalyzes the hydroxyl epimerization.Herein,we performed docking,molecular simulations,and a series of combined quantum mechanics and molecular mechanics(QM/MM)calculations to illuminate the mechanisms of two enzymes.The catalytic reactions of two enzymes occur on the quintet state surface.For SnoK,the whole reaction includes two separated hydrogen-abstraction steps and one radical addition,and the latter step is calculated to be rate limiting with an energy barrier of 21.7 kcal/mol.Residue D106 is confirmed to participate in the construction of hydrogen bond network,which plays a crucial role in positioning the bulky substrate in a specific orientation.Moreover,it is found that SnoN is only responsible for the hydrogen-abstraction of the intermediate,and no residue was suggested to be suitable for donating a hydrogen atom to the substrate radical,which further confirms the suggestion based on experiments that either a cellular reductant or another enzyme protein could donate a hydrogen atom to the substrate.Our docking results coincide with the previous structural study that the different roles of two enzymes are achieved by minor changes in the alignment of the substrates in front of the reactive ferryl-oxo species.This work highlights the reaction mechanisms catalyzed by SnoK and SnoN,which is helpful for engineering the enzymes for the biosynthesis of anthracycline nogalamycin.(3)Theoretical insight into reaction mechanism of the streptozocin synthase SznFStreptozotocin is an N-nitrosourea natural product from bacterial,which are prominent carcinogens and important cancer chemotherapeutics.Recent experiments show the complete biosynthetic pathway of streptozotocin,in which SznF enzyme catalyzes an oxidative rearrangement of the guanidine group of N?-methyl-L-arginine to generate an N-nitrosourea product.Although lots of work has been done about the structure and function of enzymes involved in the biosynthesis of streptozotocin,the catalytic mechanism of SznF enzyme is still uncertain.Based on the high-resolution crystal structure,the enzyme-O2-substrate complex models were constructed.Then,a series of combined QM/MM calculations were performed.Our results show that the substrate shows a bidentate binding to the iron center and the hexa-coordinate model of the center iron ion is obtained.The calculation results reveal two binding modes of substrate to the iron ion,which can convert each other.The spin density values show that electron transfer occurs from the substrate to dioxygen via the center iron ion,thus both substrate and dioxygen can be activated by binding to the center iron ion.The catalytic reaction occurs on the quintet state surface.A total of five steps are involved:Op abstracts the hydrogen atom from substrate,a cyclic peroxidation intermediate is formed by attacking the substrate,the formation of NO by breaking the N?-C? bond and the Op-Od bond,Fe-OH species abstracts the hydrogen atom from substrate,the substrate is attacked by NO to form an N-nitrosourea compound.The H-abstraction by Fe-OH is calculated to be rate limiting with an energy barrier of 21.0 kcal/mol.These results highlight the reaction mechanism catalyzed by SznF and provide useful information for understanding the biosynthesis of streptozotocin.(4)Insight into the catalytic mechanism of the quercetinase QueDFLAQuercetin 2,4-dioxygenase from Streptomyces sp.strain FLA(QueDFLA)is an enzyme of the monocupin family,which catalyzes the oxidative ring-cleaving reaction of quercetin using nickel ion as the active site cofactor,and the iron ion that is necessary for most dioxygenases only shows low reactivity.To understand the reaction mechanism and the activation of dioxygen by nickel ion,we performed QM/MM calculations to elucidate the reaction details and the special activation mechanism of this unique enzyme.Our calculations reveal two binding modes of dioxygen to the nickel ion,which can convert each other.Due to the overlap between the vacant d orbitals of nickel and the lone pair p orbitals of dioxygen and quercetin,electron transfer occurs from the quercetin to dioxygen via the nickel center,thus,both dioxygen and quercetin can be activated by their binding to the nickel ion.On the basis of our calculations,the triplet reactant complex favors the catalytic reaction,and the whole reaction contains four elementary steps.In particular,a nonchemical process,the Op-Od bond rotation along the nickel center,is suggested to be rate-limiting with free energy barrier of 19.9 kcal/mol.NBO analysis reveals that it the change of the coordination of Op with nickel ion that leads to the high energy barrier of this process.In general,owing to activation of substrate and dioxygen by nickel ion,the formation and collapse of the five-membered ring intermediate are quite easy,and the cleavage of O-O is in concert with the broken of two C-C bonds.Furthermore,when metal cofactor is replaced by iron ion,the rate-limiting step switches from the Op-Od bond rotation to the collapse of the five-membered ring intermediate,corresponding to free energy barrier of 30.3 kcal/mol.This study sheds insight into the reaction mechanism of QueD and contributes to our general understanding of other nickel containing enzymes.