Interaction Between The Non-conserved Amino Acid In M.grisea CYP51 Active Cavity And Its Inhibitors

Sterol 14a-demethylase is an important antifungal target which plays a key enzyme in fungal sterol biosynthesis.The researches on the structures of CYP51s and their interaction with fungicides are important for the exploitation of demethylase inhibitors(DMI fungicides,DMIs).In order to get specific and effective DMIs for M.grisea to control the rice blast,we need to investigate the characteristics of both target enzyme MG-CYP51 and inhibitor compounds by combining the molecular biology technique with computer-aided technique.In this paper,the comparison analysis was executed on two homologous 3D models of MG-CYP51 subtypes from Magnaporthe grisea and the characteristics of their interaction with diniconazole based on homology modeling and molecular docking.The results showed that both of subtypes shared the similar binding pattern with diniconazole,in which hydrophobic interaction is the most important.Diniconazole has been banded to the large hydrophobic cavity which was formed by the corresponding same or similar hydrophobic amino acids in active sites.Thus either of the two MG-CYP51 subtypes could be used as the antifungal target for designation of DMIs.In order to obtain the function of non-conservative amino acids in MG-CYP51 active cavity,after optimizing the heterogeneous expression conditions of wild-type MG CYP51 which included E.coli BL21(DE3)rosetta,temperature 18?,induction time 4h and IPTG concentration 0.4 mM.,then via site-directed mutation,we obtained 28 mutants of MG-CYP51 based on 10 acids from the F helix,SRS4 and SRS5 of MG-CYP51,followed by expression in E.coli of these mutants recombination protein.Then we compare the ability of binding to the diniconazole of these mutants recombination protein using the binding spectrum method generalized by our lab.the results showed that the change of these non-conservative acids won't hurt the structure and basic function of MG-CYP51,however,their ability binding to DMIs indicates that these acids may affect the specific binding of MG-CYP51 and DMIs,future research are needed to confirm our speculation..On the other hand,we designed 14 compounds via computer-aided drug design,The results showed ZY4 and ZY7 have the potential to be specific and eftfective lead compounds of MG-CYP51 inhibitors.Furthermore,the affinity of ZY7 for MG-CYP51 is stronger than diniconazole.The results showed the ZY7 compound could be analyzed in future research because it may serve as potential DMIs for M.grisea.