Site-directed Mutagenesis Of Penicillium Digitatum CYP51 And Screening Of New DMI Fungicides

Citrus, as the most important economic crops, whose production was reduced owing to the affecting by green mold (Penicillium digitatumGL-01), that is the most serious disease. CYP51, belong to the P450 family, are widely distributed in bacteria, fungus, animal and plant. It is responbile for catalyzing the three-step reaction of sterol 14α-demethylation and regarded as the targeted enzyme for herbicides, fungicide and drug. Inhibitiors of sterol 14a-demethylation (DMIs) are the main group of demethylase fungicides used in agriculture, such as diniconazole, triadimefon. However, the frequent usage of these compounds results in the emergement of resistance, the mechanism study of the resistance is becoming a research hotspot in agriculture and the invention of special and effective antifungal agents is of great importance.In order to study the mechanism of resistance and seek new fungicide, different mutants were constructed and their binging activities were determined by combining the molecular biology and computer-aided technology based on the Penicillium digitatum (GL-01). Besides, some potential leading compounds were screened according to antibacter activity and the binding spectra with Pd-CYP51. These results render a theoretical basis and novel ideas for special design and research of new fungicide.Homology modelling of CYP51 from P. digitatum (Pd-CYP51) formworked as the eukaryotic orthologs (the human CYP51) and the diniconazole was docking in to the activite center. Combining the conservatism of the amino acids, the speculative key amino acids 112,120, and 309 from the conservation region and nonconservation region 501 sites were selected to mutated according to the hydrophobic amino acids and amino and the size of acids side chain. The mutant activity and the binding activity were analyzed by CO difference spectrum and binding with commercialized fungicide. It is indicated that 112,120 and 309 sites were important for the bingding between the Pd-CYP51and the fungicides. However the 501 site have few influence. The mutants maintain the activity but affect the bingding with fungicides.The Kd increase and the binding activity of mutants decrease with different mutations. If they are placed by hydrophily amino acids or some amino acids which influence the substate’s access, their activity and the binding ability will be changed.Some potential leading compunds were screened by integrating the determation of antibacterial activity and the binding ability with recombinant Pd-CYP51, compared with the commercialized fungicide. It is showed that LWJ-E-13, LWJ-E-20, LWJ-E-21, LWJ-E-39, LWJ-E-41, LWJ-E-42 showed the better antibacterial activity than triadimefon with the EC50 23.43,18.23,15.84,10.26,25, and 8.33 mg/L, with Kd 3.51, 2.29,1.65,274,158,0.896μM respectively. These compounds were significantly correlated to their EC50 values. LWJ-E-42 showed the best antibacterial activity with inhibition rate 92% under the concentration of 50mg/L and better binding ability compared with thrid commercial azoles diniconazole, triadimefon, tebuconazole. It is possible to become the new potential fungicide.