Theoretical Study On Enzymatic Selectivity Mechanism And Acid Activation Mechanism Of BM2 Channel

Protein is an important component of living organisms and the main undertaker of life activities,which plays a vital role in various metabolic processes.Understanding the relationship between protein structure and function is of great significance for exploring the origin of life,revealing the evolutionary laws of organisms,and promoting the development of protein engineering,and so on.With the development of science and technology,the analysis of the structure and function of protein by experimental researches can not meet the needs of people.In recent years,computer simulation technology has become more and more mature.The application of computer simulation technology on complex biological macromolecular systems provides the accurate theoretical analysis and prediction,but also supplements the research fields that are difficult to be studied by experimental research,such as the catalytic reaction mechanism of enzymes,the allosteric mechanism of channel proteins and so on.Today,computer simulation technology has become an important research tool in the field of protein structure and function research.In this paper,the enzymatic selectivity mechanism of AGE superfamily and chorismatases,as well as the acid activation mechanism of BM2 channel were studied by molecular dynamics simulation and quantum mechanics/molecular mechanics?QM/MM?method.The main contents are as follows:1.Theoretical studies on the substrate specificity and reaction selectivity of AGE superfamilyThe AGE superfamily?AGEs?is made up of kinds of very important isomerase,which have great potential for use in the production of oligosaccharides.In this superfamily,CE subfamily catalyzes the reversible epimerization activity of the D-mannose moiety at the reducing end of oligosaccharides,while YihS subfamily shows aldose-ketose isomerase activity involved in the interconversion of monosaccharides.In order to clarify the relationship between single conserved active pocket and two activities in AGEs,we present the results for the whole process of the epimerization activity of RaCE and the isomerization activity of SeYihS by using QM/MM umbrella sampling simulations and 2D-FES calculations.Our results of MD simulations showed that RaCE and SeYihS have different substrate specificity due to the difference in the substrate access channel and the number of tryptophan residue at the active site.Moreover,our QM/MM results provide a good description of the different stages of the catalytic mechanism of two activities.For the epimerization activity catalyzed by RaCE,the whole reaction process can be divided into three stages and six steps.Our QM/MM results show that the reaction path of ring opening of RaCE and SeYihS is similar in the stage 1.The pyranose ring was opened through the way of path 1 by the His374-RaCE?His383-SeYihS?which undergoes the protonation of the O5 atom firstly,and then deprotonation of the O1 atom.Although the reaction path of ring opening of RaCE and SeYihS is similar in the stage 1,one non-conserved residue?Leu183 in RaCE,Met175 in SeYihS?will cause a small but effective change in the direction of ring opening at the stage 1.And then,this small difference can be amplified again after the step1 in the stage 2,and it will induce the catalytic mechanism to be different completely in the next second step.Finally,RaCE chooses the epimerization activity,while SeYihS prefers to the aldose-ketose isomerization activity.Therefore,we suggested that non-conserved residue group?Leu183 in RaCE,Met175 in SeYihS?which have different steric hindrance in the same position may be serve as an important factor regulating the catalytic activities in one single conserved pocket in AGEs catalysis.Our results proposed a novel viewpoint about the regulatory mechanism between CE and YihS,and it may be helpful for further experiments of rational enzyme-design based on the??/??₆-barrel basic scaffold.2.Theoretical studies on the product selectivity of chorismatasesThe metabolism of chorismate widely exists in many organisms except animals,which is essential for the survival of infectious microorganisms and plants.The pathway of chorismate metabolism have been exploited as a target for many inhibitor studies leading to herbicides or antibiotic agents.Chorismatases catalyze the cleavage of chorismate to pyruvate and different?dihydro-?benzoate derivatives.In a single conserved active site,FkbO hydrolyzes chorismate to 3,4-trans-dihydroxy-cyclohexa-1,5-dienecarboxylate?3,4-trans-CHD?whereas Hyg5 produces 3-hydroxybenzoate?3-HBA?by chorismate.In this study,we have conducted a detailed,systematic multiscale computational investigation using MD simulations and QM/MM methodology to clarify the structure-function relationship between single conserved active pocket and two reactions.The results of MD simulations showed that FkbO and Hyg5 have the minor but important difference in the substrate binding state and the water distribution at active site,which are regulated by the A/G residue group(A244^FkbO/G240^Hyg5)and the V/Q residue group(V209^FkbO/Q201^Hyg5).Our QM/MM results showed that the difference of two catalytic mechanisms is derived from the second step,which is the turning point of the two reactions.For FkbO,the glutamate would act as a general base to activate water W2 which leads to the hydrolysis reaction.But in the intramolecular reaction catalyzed by Hyg5,the glutamate would act as a general base to activate the 4-hydroxyl,forming an arene oxide intermediate.The energy barrier of the hydrolysis mechanism for FkbO and the intramolecular mechanism for Hyg5 is 20.9±0.6and 18.9±0.6 kcal/mol,respectively.More importantly,the molecular basis for the different reaction mechanisms at conserved active pocket is revealed in our study.Our results showed that the A/G residue group would cause the changes in the binding states of substrate and the orientation of the catalytic glutamate,but only these changes affect the product selectivity in chorismatases limitedly.The results of V/Q residue group mutations indicated that the distal V/Q residue group has significant impact on the selectivity of catalytic mechanisms by regulating the water properties at active site,which may be as an important factor to control the catalytic activities in chorismatases.Our results proposed a novel viewpoint about the product selectivity in chorismatases,and it may be helpful to the developement of herbicides or antibiotic agents.3.Theoretical studies on the the pH-regulated mechanism of the BM2 channelAcute respiratory infectious diseases caused by influenza viruses seriously threaten human health and cause significant economic losses.According to the World Health Organization,seasonal influenza causes 3-5 million serious cases and 250,000-500,000 deaths each year.M2 channel proteins on influenza virus envelope are activated under acidic conditions for proton transfer,playing an important role in virus replication,germination and other processes,and are a hot target for the study of anti-influenza drugs.In this study,the pH-regulated mechanism of the influenza B M2 channel?BM2?has been systematically characterized by using multiscale computer simulations.The state-of-the-art continuous constant pH molecular dynamic with replica-exchange protocol?pH-REMD?was applied to investigate the pH-dependent conformational dynamics of BM2_TM in an explicit lipid bilayer.Specifically,we determined the acid-base titration of the His19 and His27 tetrad,and the conformational transition of BM2_TM from the closed to open state as a function of pH was directly observed.We have found that the acid activation of the BM2 channel is modulated by the protonation state of His19 tetrad.The free energy change switches from slightly positive?0.2 kcal/mol?in the+2 state to negative?-0.7kcal/mol?in the+3 state,thus providing a thermodynamic mechanism for BM2activation.Furthermore,the mechanism with which His27 influences the acid activation of BM2 was characterized by classic fixed-protonation-state MD and QM/MM umbrella sampling simulations.Our simulation results show that the His19 deprotonation barrier lowers as the positive charge on the His19 tetrad increases,thus activating the channel.Our simulations identify that the second titratable histidine His27 in BM2 channel could further promote pH-mediated conformational fluctuations and speed up proton dissociation from the HxxxW motif,thus facilitating proton conduction by BM2.Finally,a unique“activation-promotion mechanism”about the BM2 proton channel is proposed,and these results may provide useful theoretical support and guidance for the experimental exploration and clinical research of influenza virus.