Study On The Interaction Between Dihydroxyacid Dehydratase And Artificial Inhibitors Related To The Design Of New Herbicide

The problem of weed resistance is becoming more and more prominent,which seriously threatens the agricultural production.Herbicides should not only consider how to kill weeds,but also take the adaptation to the environment,safety and pollution-free as the starting point,and achieve the goal of action,strong selectivity,and safety to the environment and human beings.Therefore,finding and designing new herbicides and analyzing its interaction model with the target proteins are important for environmental protection and sustainable agricultural development.Branched-chain amino acids（BCAA,including leucine,isoleucine,and valine）are essential nutrients for plant growth.The BCAA biosynthetic pathway consists of three enzymes:acetolactate synthase（ALS）,acetohydroxy acid isomeroreductase（KARI）,and dihydroxy acid dehydratase（DHAD）.Interestingly,this pathway does not exist in mammals,where they have to rely on food intake.Therefore,ALS,KARI,and DHAD are considered to be ideal targets for herbicide design.DHAD,highly conserved in different plant species,catalyzes the dehydration ofα,β-dihydroxy acids to the precursorα-keto acids of leucine,isoleucine,and valine.In the previous work,Nanjing Agricultural University designed and synthesized dozens of hit compounds on the basis of the DHAD-AA complex structure（to be published）.My master’s thesis work is to dertermine the complex structure and probe the molecular mechanism.My work will provide theoretical support for the design and optimization of new herbicides.My thesis work mainly includes three aspects:1.Purification of the target protein DHAD,the fulllength protein containing the stable[2Fe-2S]iron-sulfur cluster active center by anaerobic purification.The purity was greater than 98%.2.Finding biochemical methods to compare the activity of 11 different inhibitor molecules.The 11^#molecule inhibitor was found to have IC₅₀ value of 1.2 m M.Combined with SMT analysis technology,three new molecule inhibitors were fished.Among them,molecule H259 has higher inhibitory activity than molecule 11^#.3.Crystallization of DHAD protein and molecule H259 by co-crystallizatio and the following soaking method.The crystal structure of DHAD-H259 complex was determined at a resolution of 2.19?was obtained by analysis.The structural analysis explains why the binding force of H259 molecule and DHAD protein is not as good as that of natural product AA.According to the structural information,the molecule H259 forms a 6-coordination structure with Mg²⁺,in which the coordination distance of the carbonyl structure exceeds the normal coordination distance.The hydrophobic structure of H259 cannot be stabilized in the hydrophobic pocket,which makes the binding force inferior to natural product AA.