Study On Expression, Purification, Activation And Inhibitor Screening Of Choline Trimethylamine Lyase

Choline trimethylamine lyase(CutC)is a member of the glycyl radical enzyme(GRE)family and is widely present in human intestinal microorganisms.CutC can decompose choline,betaine and other substances in food under anaerobic conditions to produce trimethylamine(TMA).After TMA enters the human blood circulation,it will be oxidized to trimethylamine oxide(TMAO)by flavin monooxygenase in the liver).TMAO can increase the platelet reactivity in the blood and the possibility of thrombosis in the body,thus leading to cardiovascular and cerebrovascular diseases.Therefore,CutC plays a key role in the occurrence and development of cardiovascular and cerebrovascular diseases,and the study of its inhibitors can provide new solutions for the treatment of cardiovascular diseases.Our research object is choline trimethylamine lyase derived from Desulfovibrio alaskensis G20.We first constructed pET28a-CutC,pET29b-CutD,and pACYCDuet-1-isc recombinant expression vectors by molecular biology methods,and then performed expression purification to obtain CutC and CutD proteins.The optimal induction condition of CutC is 500 ?M IPTG,37 ?,4 h.The optimal induction condition for CutD was 500 ?M IPTG under strict anaerobic conditions,16 ?,14 h.Enzyme kinetic parameters are the key parameters in the study of enzyme inhibitors,and determination of CutC kinetic parameters is of great significance.We use enzyme-coupled spectrophotometric method to determine the CutC kinetic parameters,and indirectly express the amount of acetaldehyde produced by measuring the change of NADH light absorption value.We successfully activated CutC through the above experiments and constructed an enzyme-linked reaction activity measurement method.The CutC enzyme kinetic parameters were calculated,with Km value of 337.6 ?M and Vmax of 26.164 mM / min.The enzyme activity was 4.32 U,and the specific activity was 0.491 U / mg.The screening of enzyme inhibitors plays an important role in drug development.Existing CutC inhibitors have less research and lower inhibitory capacity,so we plan to screen for CutC inhibitors from various compound libraries.We adopted the method of computer virtual screening.Through the docking of a total of about 90 K compounds with MCE Fragment Library library,TCMSP compound library of Chinese medicine component small molecule library and CNS Library library,a total of more than 500 kinds of absolute values of scoring functions were selected.Compounds greater than 5,and compared with the existing inhibitors DMB and CutC,found that a variety of small molecule compounds will form hydrogen bonds with Cys489,Glu491 in CutC,and a small number of compounds also form hydrogen bonds with Tyr506.In addition,these compounds also form hydrophobic interactions with Tyr208 and Phe395.The abovementioned sites are the key sites in the binding process of choline and CutC.In addition,the free energy of binding of these compounds to CutC is greater than that of the existing inhibitors DMB and CutC.From this we have initially obtained compounds that may have a better inhibitory effect on CutC,but the specific inhibitory effect needs to be verified by subsequent experiments.