论文部分内容阅读
为阐明杀菌剂与PdCYP51的相互作用及其抗性机制,基于最新解析的真核生物人类的CYP51晶体结构,同源模建了PdCYP51的三维结构,并选取商品化的杀菌剂烯唑醇进行分子对接,预测影响PdCYP51与烯唑醇相互作用的关键氨基酸。采用定点突变技术获得了PdCYP51-Y112H、F120L、F120D和S309A 4种突变体(PdCYP51m)。结果表明,突变体蛋白PdCYP51-Y112H、F120L、F120D和S309A表达量均有不同程度的变化,PdCYP51-Y112H和F120L与未突变蛋白表达量相当,而PdCYP51-F120D和S309A表达量增加。4种突变体与杀菌剂的结合能力降低,其结合常数分别为1.28、0.18、1.03和1.31μmol/L,均大于未突变PdCYP51的0.12μmol/L。表明这些氨基酸是杀真菌剂烯唑醇与PdCYP51结合的关键氨基酸,PdCYP51与烯唑醇形成的疏水性空腔和稳定配体杀菌剂分子的作用力是酶蛋白与杀菌剂结合的重要因素。
In order to elucidate the interaction between bactericides and PdCYP51 and its resistance mechanism, the three-dimensional structure of PdCYP51 was homologously constructed based on the newly analyzed CYP51 crystal structure of human eukaryotes, and the commercial fungicide diniconazole was selected as the molecule Docking predicts the key amino acids that affect the interaction of PdCYP51 with diniconazole. Four mutants of PdCYP51-Y112H, F120L, F120D and S309A (PdCYP51m) were obtained by site-directed mutagenesis. The results showed that the expression levels of PdCYP51-Y112H, F120L, F120D and S309A varied with different degrees. The expressions of PdCYP51-Y112H and F120L were similar to those of non-mutein, while the expressions of PdCYP51-F120D and S309A were increased. The binding capacity of 4 mutants and bactericidal agents decreased with the binding constants of 1.28, 0.18, 1.03 and 1.31 μmol / L, respectively, which were all higher than 0.12 μmol / L of unmutated PdCYP51. These amino acids are the key amino acids binding fungicide diniconazole to PdCYP51. The interaction between hydrophobic cavity formed by PdCYP51 and diniconazole and the stabilizing ligand fungicidal molecule are important factors in the combination of enzyme and fungicide.