Effect Of Flavonoids Towards Biomolecules Hydroxylation Catalized By Two Dioxygenases

Methylation of biomolecules?especially primary bases and amino acids?is one of the important events of epigenetics,and its status can affect the growth,development and disease development of cells.Whereas?-KG??-ketoglutaric acid?/Fe²⁺-dependent dioxygenase catalyzes oxidative demethylation to maintain normal methylation.However,the overactivity or high-expression of dioxygenase may lead to hypo-methylation in cellular epigenetics.Therefore,dioxygenase inhibitors?and regulators?have attracted the increasing attention from researchers in recent years.Objective1.To investigate the selectivity of hydroxylation?even oxidation?of biomolecules?amino acids and primary bases?catalized by two dioxygenases?histidine demethylase JMJD3 and nucleic acid demethylase TET1?.2.To study the effect of 20 flavonoids and vitamin C towards the JMJD3-catalytic?or TET1-catalytic?hydroxylation and oxidation,and then,to discuss the possible chemical mechanisms.MethodsIn this study,JMJD3 protein and TET1 protein were selected as representatives of dioxygenase;7 bases and 20 amino acids were chosen for the substrates for the study.The 7primary bases refer to 5-methylcytosine,thymine,5-ethyluracil,theophylline,9-methylguanine,1,3-dimethylurea pyrimidine and 2-amino-4,6-dimethylpyrimidine.The 20amino acids,inducing Tyr,Phe,Met,Val,Arg,Asp,Cys,Gln,Glu,His,Ile,Gly,Asn,Leu,Lys,Pro,Ser,Thr and Ala,as substrates.In generally,the dioxygenase was mixed with substrates under the existence of?-KG/Fe²⁺.The oxidation products were further detected by the ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry?UPLC-ESI-Q-TOF-MS/MS?technology.The data were used for mass spectrum elucidation.The oxidation sites were discussed based on a chemical molecular model created using ChemBioDraw 16.0 software.In the experiment of primary bases and amino acids,phenylalanine and 5-ethyluracil were selected as substrates,flavonoids?substrate vs flavonoid=100:1,50:1 and 10:1?were added,to interfere with the oxidation reaction,the peak areas of tyrosine and 5-ethyluracil oxidation products were detected by UPLC-ESI-Q-TOF-MS/MS,to analyzed the regulation of flavonoid on catalytic oxidation of JMJD3 protein and TET1protein.Results1.In UPLC-ESI-Q-TOF-MS/MS spectra,we observed the corresponding peaks of oxidation products?C₅H₅N₃O₂,C₅H₄N₂O₃,C₆H₆N₂O₃ and C₆H₆N₂O₃?in JMJD3-mediated reaction products of 5-methylcytosine,thymine,5-ethyluracil and 1,3-dimethyluracil reaction.In addition,the corresponding hydroxylation product peaks(C₆H₉N₃O,C₇H₈N₄O_3,and C₆H₇N₅O₂)were also found in the reactions of 2-amino-4,6-dimethylpyrimidine,theophylline and 9-methylguanine.In 2-amino-4,6-dimethylpyrimidine reaction solution,both methyl groups were found to be simultaneously substituted by hydroxyl groups.In JMJD3-mediated amino acid reaction,only tyrosine,phenylalanine,tryptophan and valine were found to give rise to the corresponding hydroxylation product peaks;while methionine can be transformed the hydroxylation product under the catalysis of TET1 protein.2.When flavonoids from traditional Chinese medicines were respectively involved in the JMJD3-mediated?or TET1-mediated?hydroxylation of phenylalanine at three different molar concentrations?substrate vs flavonoids=100:1,50:1 and 10:1?,the product peak areas decreased with the increasing concentrations.However,at low concentration?substrate vs flavonoids=100:1?,the peak areas of their?isorhamnetin,dihydromyricetin,isoquercitrin and myricitrin?hydroxylated product?tyrosine?were 678,322,531 and 380,respectively.Their peak area is increased compared with the negative control group?the tyrosine peak area of the negative control group was 215?.3.The vitamin C-mediated peaks of phenylalanine hydroxylation catalyzed by the JMJD3 and TET1 enzymes were 1970 and 1751,respectively at substrate:vitamin C=10:1,and their peak area of the hydroxylated product was 10 times of negative control group.However,when 5-ethyluracil as a substrate,its catalytic activity was twice that of the negative control.Each of baicalein,chrysin,pyrogallol and scutellarein could promote the conversion of 5-ethyluracil to the JMJD3-mediated?or TET1-mediated?oxidation product.Their promotion effects were observed to depend on the different molar ratios.However,compared with the negative control group,there was no evident inhibitory effect.ConclusionsFirst,under the conditions of existence of?-KG and Fe²⁺,dioxygenases JMJD3 and TET1 can selectively catalyze the oxidation of various bases,to give rise to corresponding oxidated products.However,the oxidized products differ with the-CH₃ types:when the-CH₃ group is attached to a C atom?i.e.C-CH₃?,it usually yields a keto group;whereas the-CH₃ group is linked to a N atom?i.e.N-CH₃?,it usually produces an alcoholic-OH via hydroxylation.Second,when amino acids act as the substrate,they can be oxidized to the corresponding hydroxylated products;these amino acids include three aromatic amino acids?i.e.tyrosine,phenylalanine and tryptophan?and an aliphatic amino acid?i.e.valine?.Interestingly,the H atom of phenyl-ring is substituted by-OH,and phenylalanine is correspondingly oxidized to tyrosine.This may provide a new pathway for phenylalanine to transform into tyrosine.Third,such dioxygenases-catalytic effect can be affected by Vitamin C and natural flavonoids.Vitamin C can promote JMJD3 and TET1 to catalyze the oxidation of various substrates.It may be associated with pro-oxidant action,rather than its specific TET1 protein-binding.Last but not least,most flavonoids can inhibit JMJD3-catalytic?or TET1-catalytic?base oxidation and amino acid hydroxylation catalyzed in a dose-dependent manner at experimental concentrations.However,some flavonoids?such as dihydromyricetin,myricitrin,isorhamnetin,and so on?are able to regulate the dioxygenases-catalytic hydroxylation of substrates at different ways:flavonoids at low concentration can promote the dioxygenases-catalytic oxidation;however,flavonoids at high concentration can inhibit the dioxygenases-catalytic oxidation of substrate.These multiple regulation ways may be related to the ability of Fe²⁺-complexing and the pro-oxidation of phenolic-OH?especially the pyrogallol moiety?in flavonoids.In general,Chinese herbal flavonoids regulate the catalytic oxidation of dioxygenase may be through the removal of the generated free radicals;complexing Fe²⁺to reduce Fe²⁺content in solution;reduced ferric oxide Fe^VI=O,lower its valence,make it lose its catalytic activity;or occupy the empty space of the enzyme molecules to prevent the mosaic of Fe^VI=O for inhibiting its catalytic activity.However,some flavonoids also exhibit pro-oxidation at specific concentrations,which may be one of the reasons why some flavonoids can enhance enzymatic oxidation or hydroxylation.