基于柑橘绿霉菌CYP51同源模建的定点突变与抗性机制分析

Site-directed mutagenesis and resistant analysis based on the homology modeling of Penicillium digitatum CYP51

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。

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 observations suggest that three sites are key amino acids,and hydrophobic cavity and stable force is the important factors in binding between PdCYP51 and diniconazole.