| Enzymes are a kind of extremely important biological catalysts.It is because of the action of enzymes that chemical reactions in organisms can be efficiently and specifically carried out under extremely mild conditions,and constitute an important basis for all life processes.The enzymes are proteins and a small amount of RNA in chemical nature,and there are many types of enzymes due to their different structures and catalyzed reactions.About one-third of enzymes in nature require some metal ions such as iron,molybdenum,copper,zinc,etc.as cofactors or activators,and these enzymes are called metalloenzymes,of which iron-containing metalloenzymes are the most common and diverse types of enzymes.According to the difference in the number of iron ions,the types of ligands,and the coordination structure in the active center of iron-containing enzymes,iron-containing enzymes can be divided into various types,such as heme enzymes and non-heme enzymes.With the increasing application of enzyme-catalyzed reactions in medicine,food,textile and other fields,synthetic biology and enzyme engineering technology have become one of the hot issues in the field of biological science and technology,and the analysis of enzyme-catalyzed reaction mechanism has become an important basic science problem in the field of biosynthesis.Resolving the mechanism of enzyme-catalyzed reactions at the atomic level is full of challenges for both experimental studies and computational simulations,mainly due to the complexity of enzyme-catalyzed reactions.At present,the combination of experiment and theoretical calculation is mainly used to study the mechanism of enzyme-catalyzed reaction.The information on the crystal structure of enzymes,the binding state of substrates,reaction rates and certain reaction intermediates can be obtained through experiments,while the structural and energetic information on the transition states and intermediates of each substrate reaction involved in enzyme-catalyzed reactions can be obtained through theoretical simulations,and the close combination of the two enables the microscopic mechanisms of many enzyme-catalyzed reactions to be resolved.In recent years,experimental scientists have obtained more and more crystal structures of metalloenzymes,which have laid a good modeling foundation for the analysis of their catalytic reaction mechanisms.In this work,we have mainly used molecular dynamics simulations and quantum-mechanical/molecular-mechanical(QM/MM)calculations to perform a systematic theoretical study of two iron-containing metallooxygenases(cytochrome P450 enzyme TleB and non-heme dioxygenase NicX),revealing the most probable pathways of enzyme-catalyzed reactions at the microscopic level,elucidating the structural evolution and electron transfer of the species involved in the reaction process and explaining the different roles played by some key residues in the catalytic reactions.Our calculation results not only analyze the mechanism of the two enzyme-catalyzed reactions at the atomic level,but also provide some theoretical guidance for the experimental research and enzyme design of related enzymes.The main research works of this dissertation are as followed:(1)Mechanistic Insights into the P450 TleB-Catalyzed Unusual Intramolecular C-N Bond Formation Involved in the Biosynthesis ofIndolactam VIndolactam V,a known biosynthetic precursor of indolactam alkaloids,is the main pharmacophore of this family and exhibits potential PKC(protein kinase C)activation.A key step in indolactam V biosynthesis is the formation of indole-fused nine-membered lactam core by intramolecular C-N bond formation.In this work,we report a theoretical study of the unique cytochromes P450 TleB enzyme-catalyzed direct and selective C-H bond amination reaction that can generate indolactam V from the dipeptide N-methylvalyl-tryptophanol.By performing molecular dynamics simulations and quantum-mechanical/molecular-mechanical(QM/MM)calculations,we revealed that the C-H bond amination involves one step of proton transfer from the N1-H of indole to Fe(Ⅳ)=O,one step of hydrogen abstraction of N13-H in substrate side chain by Fe(Ⅳ)-OH,and the diradical coupling,in which two conformational changes of the side chain of substrate are necessary.In the enzyme-substrate complex of TleB,the N-H of indole ring of substrate forms a strong hydrogen bond with the Fe(Ⅳ)=O in compound I,and the porphyrin radical cation accepts an electron from the substrate to form the closed-shell electronic configuration.Thus,the compound I in the enzyme-substrate complex can not be described as Fe(Ⅳ)=O coupled to a porphyrin radical cation,which is different from those of other P450 enzymes.Besides,two stages of conformational changes of the substrate side chains correspond to energy barriers of 10-12 kcal/mol.From the structure point of view,it is the rotatable long side chain of substrate and large flexible active pocket of TleB that make the intramolecular diradical coupling feasible.Our findings may provide useful information for further understanding the Tleb-catalyzed intramolecular C-H bond amination and other bio-catalyzed intramolecular diradical coupling.(2)Mechanistic Insights into Pyridine Ring Degradation Catalyzed by 2,5-Dihydroxypyridine Dioxygenase NicX2,5-Dihydroxypyridine dioxygenase(NicX)from Pseudomonas putida KT2440 is a mononuclear non-heme iron oxygenase that can catalyze the oxidative pyridine ring cleavage.Recently,the reported crystal structure of NicX lends support to an apical dioxygen catalytic mechanism,while the mechanistic details remain unclear.In this work,we constructed the Fe(Ⅱ)-O2-substrate complex model and performed a series of combined quantum mechanics/molecular mechanics(QM/MM)calculations to illuminate the catalysis of NicX.Our results reveals that,although the substrate does not directly coordinate with the central iron ion,there is an electron transfer from the substrate to the Fe-coordinated dioxygen,and the active form of reactant complex can be described as DHP’+-Fe(Ⅱ)-O2·-,which is different from other similar mononuclear non-heme iron.The NicX-catalyzed pyridine ring degradation contains three parts,including the attack of Fe(Ⅱ)-superoxo on the activated pyridine ring,the dissociation of Op-Od bond,and the ring opening of the seven-membered-ring lactone.Owing to the radical characteristic of pyridine ring,the first attack of Fe(Ⅱ)-superoxo on the C6 of pyridine ring was calculated to be quite easy.In the second step reaction,the dissociation of Op-Od bond leads to the incorporation of the first oxygen atom into the substrate,which is the rate-limiting step of the overall reaction with an energy barrier of 18.0 kcal/mol.The resultant intermediate then undergoes an arrangement by the intramolecular attack of Od’ to the carbonyl C5,forming the seven-membered-ring lactone.Finally,the Fe(Ⅲ)-oxo attacks the carbonyl C5 of lactone,accompanied by the ring opening,to generate the N-formylmaleamic acid.His105 can promote the reactivity by denoting a proton to Fe(Ⅲ)-oxo,but it is not a necessary residue.Besides the ligated residues of iron,other pocket residues such as Glu177,His 189 and His 105 mainly play roles in anchoring the substrate. |
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