稻瘟菌CYP51蛋白F螺旋区保守氨基酸残基与

Interaction between the conserved amino acid in

摘要:

【目的】研究稻瘟菌CYP51蛋白F螺旋区保守氨基酸残基与烯唑醇的相互作用机制，为稻瘟病菌新型高效特异杀菌剂的开发提供理论依据。【方法】设计稻瘟菌CYP51蛋白F螺旋区保守氨基酸残基突变体P222C、P222H、I223A、I223W、N224A、N224S，截去N端跨膜区36个氨基酸后，在大肠杆菌BL21(DE3) Rosetta菌株中过量表达，采用结合光谱法分析诱导蛋白对烯唑醇的结合能力。【结果】表达的目标蛋白均保持了对药物的结合能力，呈现出II型的结合光谱曲线。相对于野生型蛋白，突变体I223W和I223A对烯唑醇的结合常数Kd值基本不变，N224S、N224A、P222C的Kd值都略有增大，无显著性差异(P>0.05)，但是，P222H的Kd值有了显著的增大(P<0.05)，表明突变体P222H对烯唑醇的亲和能力显著降低。【结论】稻瘟菌CYP51 P222位点的疏水性与药物结合密切相关。

Abstract:

[Objective] The interaction between the conserved amino acid in Magnaporthe oryzae CYP51 F helix and Diniconazole was studied, aiming to help for designation of new specific and effective demethylase inhibitors for M. oryzae. [Methods] Six mutants (P222C, P222H, I223A, I223W, N224A, N224S) of M. oryzae sterol 14α-demethylase (MGCYP51) F helix were constructed with truncation of N-terminal 36 residues and heterologously expressed in Escherichia coli BL21(DE3) Rosetta. The binding ability of the recombination proteins to the diniconazole was detected by using the binding spectrum method. [Results] All of the recombination proteins had the activity to binding to the diniconazole and presented type II spectrum. Compared with the wild type protein, the Kd values of the mutations I223W and I223A binding to diniconazole were essentially unchanged, and the Kd values of N224S, N224A, P222C increased slightly with no significant difference (P>0.05), while the Kd of P222H increased significantly (P<0.05), indicating that the ability of the mutation P222H binding to the diniconazole significantly reduced. [Conclusion] The hydrophobic of the site P222 could play a major role in the binding of MGCYP51 to the diniconazole.