Application Of Binding Spectra To Analysis Recombinant Penicillium Digitatum Cyp51 And Its Mutants With Fungicides

Citrus green mold(Penicillium digitatum) is one of the most important fungal pathogens that can cause disease of citrus.Large numbers of rotting oranges will be triggered by Citrus green mold,it's very significant that a huge loss of life and productiong caused by Citrus green mold.Sterol 14a-demethylase(CYP51) is the only member of the cytochrome P450 superfamily widely present in bacteria,fungi,plants and animals.In all cases it catalyzes a three-step reaction of sterol 14a-demethylation. Inhibitiors of sterol 14α-demethylation(DMIs) are the main group of fungicides used in agriculture.As representatives of triazole fungicides,diniconazole and triadimefon are widely used in storage of citrus due to the characteristics that it can protect and control at the same time,applying of low dosage with a long period of efficacy.Inhibition of sterol 14a-demethylation by DMI fungicides result in the depletion of ergosterol and accumulation of abnormal sterol intermediates.Consequently the growth of fungi was stopped.However,triazole fungicides has many defects such as site-specific and resistance nowdays.With the rapid increase in the incidence of fungal diseases in agriculture,as well as the emerging of DMIs-resistant strains,there is an urgent need to develop a new generation of azole fungicides with high efficiency,low toxicity and highly specific.In order to provide a more effective way for the development of fungicide,we have investigated the characteristics of both the antifungal activities of three new nitrogen-containing compounds and inhibitor compounds by combing the molecular biology technique with computer-aided technique.According to bioinformatics and the results of molecular docking,we have built several different P.digitatum CYP51 mutants, and analysised the combination capacity between mutants and compounds.The results obtained in this study. 1.The binding capacity of diniconazole,triadimefon and four kinds of Q seriers compouds with CYP51 have analysised using heterologous expression P.digitatum CYP51.The effects of enzyme concentration on the binding spectra were investigated. The antifungal activities of all compounds were evaluated too.The results showed that proper concentration were necessary for obtaining accurate binding spectra.Several poteintial effective and selective lead compounds were obtained,including C-3,C-5 and C-12 in XF seriers;Z-5 in ZST seriers;Q-6,Q-9 and Q-10 in Q seriers.Compared with triadimenol and triadimefon,the effects were equivalent or better.These seven compounds have great potential for development. 2.According to bioinformatics and the results of molecular docking,several different P.digitatum CYP51 mutants have constructed) including mutants that truncated 18 N-terminal amimo acids and only contained 272 amino acids near the active site. Analysised the combination capacity between mutants and compounds,the results showed that the Kd of CYP51 with C seriers compounds was similar to CYP51Pd-18. Enzyme activity was well preserved.This is in accordance with the contrast results of three-dimensional structure between full-length CYP51 and CYP51Pd-18.