Nucleic Acid Bases Hydroxylation Mechanisms Catalyzed By TET1 And Regulation Of Small Molecules Of Tradition Chinese Herb

ObjectiveThe present study focused on systematically discussing chemically viable mechanisms for TET1 demethylation,and furture screening tradition Chinese medicine to disscuss these regulation function.MethodsIn this study,TET1,a Fe2+/2-KG-dependent dioxygenase,was used as the catalytical enzyme for the investigation,and 5mC,benzene,toluene,DMSO,and d6-DMSO were ultilized as the substrates.The reaction was conducted in H2O18,D2O solution respectively while O2 and 18O2 were blown into the reaction system.The reaction products(especially 5hmC)were then demtermined using GC-MS or UPLC-MS,to investigate the hydroxylation mechanism by the TET1 enzyme.Subsequently,Proanthocyanidin,wedelolactone,salvianolic acid B,baicalin,quercetin-3-rhamnoside,7,8-dihydroxyflavone,luteolin,cyaniding chloride,taxifolin were added into the reaction mixture,to observe the change of 5hmC levels.However,for the convenience of determination,the reaction products were derived using MSTFA in GC-MS analysis.ResultsThe data from GC-MS or UPLC-MS indicated that TET1 could catalyze 5mC to form 5hmC.Benzene was found to give no phenol product(molecular ion peak m/z 94).However,toluene,DMSO,d6-DMSO were hydroxylated by TET1 enzyme,and gave molecular ion peak were at m/z 108,m/z 94,m/z100,respectively,C5H9N3O2 was detected in the reaction system that TET1 react with 5mC in H2O18,18O2.However,C5H8N3O2 was not present in the D2O reaction system.When proanthocyanidin,wedelolactone,salvianolic acid B,baicalin,quercetin-3-rhamnoside,7,8-dihydroxyflavone,luteolin,cyaniding chloride and taxifolin were added into the reaction mixture,the yield peak areas of 5hmC were calculated as 1053811,842578,794099,1277581,640249,555535,408748,0,respectively.ConclusionIt can be concluded that 5mC can be converted to 5hmC in vitro by TET1 enzyme.This process is initiated by hydrogen abstraction from the methyl group,followed by a radical rebound hydroxylation of the methylene group.Nevertheless,in this raction,O atom comes from O2,H2O.Nine small molecules from traditional Chinese herbal medicines(i.e.proanthocyanidin,wedelolactone,salvianolic acid B,baicalin,quercetin-3-rhamnoside,7,8-dihydroxyflavone,luteolin,cyaniding chloride and taxifolin)more or less reduce the reacted hydroxylation level of 5mC.It suggests that these small molecules may be potential inhibitors of TET1 enzyme in the process of hydroxylation of 5mC.