Crystal Structure Of Flavonoid C-glycosyltransferase In Brachypdium Distachyon

Flavonoid is a kind of polyphenols compound that exists extensively in plants.It has attracted a wide spread attention because of its positive function in nutrition and clinic.Lipophilic micromolecule glycosylation that happens in plants is to adapt the circumstance better and gain more opportunities to survive.Flavonoid glycosylation is a mechanism that protect plants from inbreak and store bioactivator in vacuole.Usually,Glycosyltransferases(GTs)are responsible for flavonoid glycosylation.There are some differences between the process of O-glycosylation and C-glycosylation.Flavonoid O-glycosylation is attaching glycosyl to the O-3 or O-7 position of flavonoid directly.While the research in buckwheat showed that the flavonoid C-glycosylation needed 2-hydroxyflavonone as the acceptor substrate to connect the glycosyl to flavonoid C-6 or C-8 and the process was catalyzed by CGT after the treatment of P450,and the dehydration reaction was the last step of C-glycosyltransferase.So far,there are only six flavonoid OGTs in plant have determined the crystal structures,which are from medicago,grape,clitoria ternatea and Arabidopsis thaliana.It is known that OGT and CGT function different,but the reason is unclear.The function of a protein is determined by its structure.Only determine the CGT structure with substrate can explain the CGT catalytic mechanism in molecular level,and to understand the functional differences between CGT and OGT.In this study,we chose OsCGT1,OsCGT2,OsCGT3 from Oryza sativa and BdCGT1,BdCGT2 from Brachypodium distachyum as the research of C-glycosyltransferase.And we constructed the express vector using pET-28b-sumo,pET-28b-3C,pGEX-6p-1 and did the expression,purification and crystallization of OsCGT1,OsCGT2,OsCGT3,BdCGTl,BdCGT2.The result showed that sumo-OsCGTl,sumo-OsCGT2,sumo-OsCGT3,sumo-BdCGT1 and 6*His-BdCGT2 were avalible.We used the Crystal Screening Kit from Hampton Resaerch and Emerald BioSystems to obtain crystal conditions of OsCGTl,OsCGT2,OsCGT3,BdCGT1 and BdCGT2.We got the crystal of BdCGTl,BdCGT2,but failed on OsCGT1,OsCGT2,OsCGT3 crystallization.We determined the crystal structure of BdCGT2 at 2.5 A resolution.We aligned the five kinds of CGTs with other OGTs and CGTs,making a hypothesis that some residues were the key residues for C-glycosylation.It is the base for further study of CGT's structure,function catalyzing mechanism and gene modification.