Drug Screening Based On DapE Protein Structure

Rice bacterial blight is a bacterial disease caused by infection with Xanthomonas oryzae pv.oryzae,which causes white spots on rice leaves.In the 1980s,the disease caused the loss of more than 500,000 catties of rice in Yongxiu County,Jiangxi Province.The control of rice bacterial blight is mainly based on chemical control,but Xanthomonas oryzae will gradually develop resistance to the currently used drugs.Therefore,it is imminent to develop some new,targeted chemical drugs with good antibacterial effect.N-succinyl-L,L-diaminopimelic acid desuccinylase（dap E-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase,Dap E）is a bacterial lysine/diaminopimelic acid biosynthetic pathway.The key enzyme exists in all gram-negative and most gram-positive bacteria.Its deletion will affect the formation of bacterial normal cell wall,resμLting in bacterial non proliferation and bacterial death.Mammals do not synthesize lysine by themselves through external intake,and lysine synthesis in plants and fungi does not depend on Dap E.Therefore,Dap E is a good target against bacterial diseases and can be used in the study of bacterial blight disease in rice.In this thesis,a highly reliable Xanthomonas oryzae Dap E（Xoo Dap E）protein was obtained by Alphafold modeling method.After virtual screening of Zinc small molecμLe database,Colistimethatesodium（CMS）was founded.And the compound 4-oxa-4,5,6,7-tetrahydrobenzo[B]furan-3-carboxylic acid（TBFC）,also were found to bind well to proteins.Through a series of operations such as plasmid transfection,protein induction and expression,extraction and purification,the Xoo Dap E protein with a purity greater than 95%and no aggregation and denaturation was obtained.Through isothermal calorimetry（ITC）,it was found that CMS was bound to the protein.Exothermic,the binding force KD is 4.69×10^-5,and the binding of TBFC to the protein showed no reaction heat and affinity.Using liquid antibacterial method,it was found that CMS can have a good inhibitory effect on Xanthomonas oryzae at 3.2μmol/L.In the in vivo experiment of rice,it was found that the preventive effect of CMS and TBFC on rice bacterial blight disease was greater than that of treatment effect.Finally,the composite crystals of Xoo Dap E protein and small molecμLes were screened,and the effects of protein concentration,small molecμLe concentration,type and concentration of precipitant,p H,ion concentration,temperature and other factors on protein crystallization were tested.After collecting Xoo Dap E protein crystals and small molecμLe-protein complex crystals,X-ray diffraction was used to obtain the real protein structure and protein-small molecμLe co-crystal structure data.In conclusion,CMS has a significant inhibitory effect on Xanthomonas oryzae,and can be used as a preventive and therapeutic drug for rice bacterial blight.TBFC has a certain effect on Xanthomonas oryzae,but the effect is not strong,and it can be used as a lead compound for further transformation.