Theoretical Studies On The Catalytic Mechanisms Of Several Metal-containing Enzymes And Glycosidases

Enzymes are essential to all metabolic processes.Due to the catalysis of the enzymes,the metabolic rates can be fast enough to sustain life activities.The high efficiency and selectivity together with the great variety of reaction have made the industrial application of enzymes a heated research point.Many enzymes have been used in environmental,industrial and medical areas.With the development of enzymology,there must be much more engineered enzymes and biomimetic catalyst to be taken into applications.Fully understanding the activities and specificities of enzymes are not only foundational in biology,but also the precondition of enzyme engineering and drug discovery.Because of the complexity of enzymes and the reaction they catalyzed,it is difficult to get a full view of them by experiments alone,and it is much more difficult to observe all the intermediate and transition states involved in those reactions.In recent years it becomes popular to use computational modeling and simulation to get a detailed and atomic-level view into the dynamics and reactions of biomolecules.In computational enzymology,we need to choose appropriate method according to the questions to be involved.To fully understanding the catalytic mechanism of several metal-containing enzymes and glycosidase,the combined quantum mechanics/molecular mechanics(QM/MM)method was employed in this dissertation.Based on our calculation results,the optimal reaction mechanisms of these enzymes have been determined,the reaction details have been delineated,and the important roles of some active site residues have been identified.These results are in well agreement with corresponding experimental observations,which give reasonable explanation to some experimental results,and provide supplement information to experiments.The main contents are listed as follow:(1)Mechanistic Insights into the Decoupled Desaturation and Epoxidation Catalyzed by Dioxygenase AsqJ in the Biosynthesis of Quinolone Alkaloid.The quinolone scaffold plays an important role in building chemical libraries of bioactive compounds.The pharmaceutically attractive scaffold of 4’-methoxyviridicatin,4-arylquinolin-2(1H)-one,has been found in a variety of quinolone and quinolinone alkaloids.AsqJ from Asprgillus nidulans is a non-heme FeⅡ/α-ketoglutarate-dependent dioxygenase that catalyzes the conversion of benzodiazepinedione into 4’-methoxyviridicatin involved in the biosynthesis of quinolone alkaloid.Recently,the crystal structures of AsqJ in complex with the substrate and intermediate for mimicking the various stages of the reaction cycle have been reported,and a series of experiments have demonstrated that AsqJ performs the decoupled desaturation and epoxidation reactions.Herein,on the basis of the crystal structures of AsqJ,we employed the QM/MM approach to explore both the desaturation and epoxidation processes catalyzed by AsqJ.Optimized structure of the active site reveals that the FeⅣ-oxo should firstly undergo an isomerization to initiate the reaction.In the desaturation process to create the double bond,the abstraction of the first hydrogen follows the a-channel mechanism,which is the rate-limiting step,corresponding to an energy barrier of 19.3 kcal/mol.The abstraction of the second hydrogen is calculated to be quite easy.After the desaturation process,the regenerated FeⅣ-oxo species attack the C=C bond of the desaturated intermediate to initiate the epoxidation reaction,corresponds to an energy barrier 18.1 kcal/mol.Besides,calculations using the substrate without N4-methyl reveal that the H-abstraction by FeⅣ-oxo species is still accessible,which suggests that the absence of N4-methyl does not affect the desaturation process itself,but may influence the other processes which are prior the desaturation process.Our results clarified the detailed catalytic mechanism of AsqJ and illustrated the residue role of active site,which can provide useful information for understanding the biosynthesis of quinolone and quinolinone alkaloids.(2)Insights into the catalytic mechanism of imidazolonepropionase(HutI)from Bacillus subilis:A QM/MM studyThe degradation of histidine is tightly regulated in the cell.Imidazolonepropionase is the third enzyme in the universal histidine degradation pathway,it catalyzes the hydrolytic cleavage of carbon-nitrogen bond of 4-imidazolone-5-propionic acid to yield L-formiminoglutamic acid.In this paper,the catalytic mechanism of HutI from Bacillus subtilis has been studied using a combined quantum mechanics and molecular mechanics(QM/MM)approach.The reaction details have been delineated at the atomistic level.Our calculations reveal that the activation of hydrolytic water is prior to the binding of the substrate.The residue E252 acts as the general base to abstract the proton of the Zn2-coordinated water molecule through a water bridge,and the activation energy for this process is calculated to be 6.4 kcal/mol.After the binding of the substrate,the bridging water between Zn2+-coordinated water molecule and E252 is replaced by the substrate,while the residues D324 and H272 can hardly activate the Zn2+-coordinated water molecule.Based on our calculation results,the stereoselectivity of the enzyme determined in the substrate binding step and the first catalytic step for the two isomers of(S)-IPA respectively.These results presented here provide insightful information for fully understanding the catalytic mechanism of HutI.(3)Insights into the Catalytic Mechanism of N-Acetylglucosaminidase Glycoside Hydrolase from Bacillus subtilis:A QM/MM StudyThe peptidoglycan(PG)metabolic process is essential for bacterial growth.β-N-acetyl-glucosaminidases(NagZ enzymes)are involved in PG process and catalyze the removal of terminal N-acetylglucosamine in PG fragments.According to the amino acid sequence and secondary structures,NagZ enzymes should belong to the glycoside hydrolase family GH3.However,recent experimental study revealed that NagZ enzymes are glycoside phosphorylases rather than glycoside hydrolases.To further understand the catalytic process of NagZs at atomistic level,the reaction mechanism of NagZ from Bacillus subtilis(BsNagZ)has been studied by using a QM/MM approach.Our calculation results show that the glycosylation of the substrate is the rate limiting step of the whole catalytic cycle with an energy barrier of 19.3 kcal/mol,which is close to the free energy barrier(16.4 kcal/mol)estimated from the experimental rate constant.For deglycosylation,both the hydrolysis and phosphorylation of the glycosyl-enzyme intermediate were explored.The phosphorylation corresponds to the lower energy barrier than hydrolysis(1.8 vs 17.7 kcal/mol),giving theoretical support to the previously suggested phosphorylase activity of NagZ enzymes.In both the glycosylation and deglycosylation steps,the oxocarbenium-ion-like transition states are always involved,and the substrate distortion in the active site can significantly facilitate the reaction,in which residue D123 plays a key role for this distortion.This is the first computational report for understanding the phosphorylase activity of NagZ enzymes,and can aid the development of inhibitor aimed at these enzymes.(4)A QM/MM Study of the Catalytic Mechanism of a-1,4-Glucan Lyase from the Red Seaweed Gracilariopsis lemaneiformisα-1,4-Glucan lyase(GLase,EC 4.2.2.13),a unique glycoside hydrolase family member,specifically cleaves the α-1,4-glucosidic linkages in glycogen,starch and malto-oligosaccharides to produce 1,5-anhydro-D-fructose from the non-reducing end.Previous studies have proved that GLase belongs to the retaining glycoside lyase,and the catalytic reaction contains both the glycosylation and deglycosylation/elimination steps,in which a covalent glycosyl-enzyme intermediate is involved.On the basis of the newly reported crystal structure of GLase(2X21)and the speculated mechanism,the whole catalytic cycle of GLase has been studied by using QM/MM method.Calculation results indicate that the whole catalytic cycle contains five elementary steps.Firstly,the aspartic acid residue D665 acts as acid to protonate the glycoside oxygen,which is concerted with the cleavage of glycoside bond.Then,the residue D553 functions as the nucleophile to attack the anomeric carbon to form the glycosyl-enzyme intermediate.Different from the retaining α-glucosidases whose glycosylation is typical concerted process,the glycosylation process of Glycosidic lyase follows a stepwise mechanism.For the deglycosylation/elimination step,two cases with or without the maltotriose group in the active site were considerated.The departure of the maltotriose can facilitate the proceeding of this process.The deprotonated aspartic acid residue D553 further acts as a catalytic base to abstract the C2-proton of the glucosyl residue.The proton abstraction in the deglycosylation/elimination step is calculated to be the rate-limiting step of the whole catalytic reaction,which corresponds to the energy barriers of 20.69 and 18.53 kcal/mol for both of the two cases.