The Proteomic Characteristics,regulatory Enzymes And Functions Of Lysine 2-hydroxyisobutyrylation In Prokaryotes

A great variety of protein post-translational modifications(PTMs)are dynamic changes under the control of enzymes and participate in the regulation of various physiological and pathological processes,which is of great biological significance.In recent years,the discovery of new lysine modifications have attracted much attention,among which lysine-2-hydroxyisobutyryl(Khib)is a new type of histone acylation modification recently reported.The research shows that the occurrence of this modification on human histone can regulate gene transcription,and the occurrence on yeast histone is related to glucose homeostasis,suggesting that Khib plays an important role in eukaryotes.However,does the modification exist in prokaryotes? And its biological function and effect on prokaryotes are both still not clear so far.Based on this,here we systematically investigate the distribution,biological function and regulatory mechanism of Khib in prokaryotes.Firstly,the presence of Khib in prokaryotes including E.coli and P.mirabilis was found by western blot assay.Then,4735 modified sites were identified on 1051 proteins of P.mirabilis by combining affinity enrichment with mass spectrometry based proteomics,and thus the distribution,molecular network and pathway of Khib were analyzed by bioinformatics.According to our best knowledge,it is the first time for the discovery and systematic characterization of Khib in prokaryotes.Further studies show that Khib is widely distributed and many of them are located in important protein functional domains,indicating its potential function for regulating substrate proteins.Among them,Khib on metabolic regulatory enzyme is the most abundant.Through in vitro experiments,we have proved that Khib on K343 site of enolase(ENO)and K18 site of diphosphoglyceride mutase(GPMA)reduce the enzyme activity of substrate,and through the calculation of molecular docking,we have further proved that the occurrence of modification will make the substrate molecule(2PG)far away from the active sites of metabolic enzyme(ENO and GPMA),which results in the change of binding between protease and substrate.At the same time,we also proved that different carbon metabolism can regulate the level of Khib in vivo.So far,our study shows that this modification is dependent on metabolism and has regulatory function in cell metabolism.In order to further reveal the regulatory mechanism of Khib,we carried out the study of de-2-hydroxyisobutyrylase.First of all,we combined our recently developed ‘new analysis method based on multivalent affinity photo-crosslinking for binding proteins of lysine PTM’ with quantitative proteomics technology to screen the specific binding protein of Khib,and identified Cob B as a potential de-2-hydroxyisobutyrylase.Next,we constructed the bacterial systems of overexpression and knockout of Cob B,respectively,and demonstrated that Cob B has the ability to remove Khib in the bacterial from in vivo experiment;at the same time,we proved that Cob B can remove Khib at the protein and peptide level through in vitro experiments,and further studied the influence of its active sites,enzyme inhibitor.The enzyme activity were characterized by kinetic and thermodynamics experiments.And therefore,Cob B serving as a de-2-hydroxyisobutyrylase in prokaryote was found and proved for the first time.On this basis,we found that R58 of Cob B is the key site of its Khib removal activity.Furthermore,99 significant regulatory Khib sites targeted by Cob B were identified by quantitative proteomics and thus the molecular network of Khib regulated by Cob B was built by the identification of over 5799 Khib sites.Finally,through a series of biochemical and molecular biological experiments,we showed that Cob B can enhance the activity of ENO by removing the metabolic enzyme ENO-K343 hib,and then change the metabolism and affect the molecular mechanism of bacterial growth.In the aspect of 2-hydroxyisobutyrylase,we constructed the over expression system of acetylase of 18 GNAT families in E.coli by structural analysis and enzyme activity prediction,and preliminarily screened the 2-hydroxyisobutyrylase by immunoblotting.Thus Ypfi was selected as a potential modifying enzyme of Khib.Next,we constructed the Ypfi knockout system of E.coli,and proved that Ypfi is the 2-hydroxyisobutyrylase in vivo by western blot assay.Furthermore,467 significant regulatory Khib sites targeted by Ypfi were identified by quantitative proteomics and thus the molecular network of Khib regulated by Ypfi was built by the quantification of over 4351 Khib sites.It was further verified in vitro through the modification reaction experiment on protein level.In vitro experiments,we found that the 2-hydroxyisobutyrylase function of Ypfi depends on ATP,and R319 site affects the binding of Ypfi to ATP,and then affects the modifying enzyme activity of Ypfi.We found that Ypfi can specifically catalyze the 2-hydroxyisobutyrylation of K121 site of H-NS,a key histone like protein in E.coli.At the same time,we found that the 2-hydroxyisobutyrylation of H-NS K121 site can affect the binding of H-NS to gene.Furthermore,the EMSA experiment after the reaction of Ypfi with H-NS demonstrated that Ypfi can mediate the 2-hydroxyisobutyrylation of H-NS K121 site in vitro to influence the molecular mechanism of H-NS binding to DNA.In conclusion,Khib has been found in prokaryotes for the first time by proteomics and biochemical methods in this study.We further analyzed its distribution characteristics,identified and verified its regulatory enzymes,and analyzed its regulatory function on metabolism and transcription of prokaryotes.