Abstract:Penicillium digitatum sterol 14α methylation enzyme (PdCYP51), the targeted enzyme of azole fungicides, was widely applied in preventing the disease caused by P.digitatum. To elucidate the mechanism of interaction between the PdCYP51 and fungicides, 3D-structure of PdCYP51 was built based on the eukaryotes human CYP51 crystal structure, and commercial diniconazole was docked into the active cavity of PdCYP51. The key amino acids which influence the interaction between the PdCYP51 and diniconazole were predicted, and the mutants (PdCYP51-Y112H, F120L, F120D and S309A) were obtained by site-directed mutagenesis. The results indicated that the expression of mutants changed differently and PdCYP51-Y112H and F120L mutants keep the same amount with PdCYP51; while PdCYP51-F120D and S309A increased. Compared with the wide PdCYP51 (0.12 μmol/L), the affinity Kd values of these four mutants against fungicide determined by spectral analysis increased, with 1.28, 0.18, 1.03, 1.31 μmol/L, respectively, and their binding activity with diniconazole reduced. These observa- tions suggest that three sites are key amino acids, and hydrophobic cavity and stable force is the impor- tant factors in binding between PdCYP51 and diniconazole.