点突变对结核分支杆菌KatG蛋白活性和结构的影响

Effect of Missense Mutations on the Structure and Function of the Catalase/Peroxidase, KatG,from Mycobacterium tuberculosis

目的 探讨点突变对结核分支杆菌KatG蛋白过氧化氢-过氧化物酶活性和蛋白结构的影响,深入研究INH耐药的发生机制。方法 采用三步法PCR对KatG基因的4个位点进行定点诱变,在大肠埃希菌BL21体系中表达和纯化相应诱变蛋白,并测定其过氧化氢酶、过氧化物酶活性、二硫键的形成及荧光光谱变化,最后利用酵母细胞色素C氧化酶模型进一步探讨点突变对KatG蛋白过氧化氢-过氧化物酶活性和结构的影响。结果 完成KatG基因4个位点的诱变及相应诱变蛋白的表达和纯化。过氧化氢酶、过氧化物酶活性测定发现3个诱变蛋白[KatG(W321G)、KatG(R418Q)和KatG(A456S)]活性基本丧失,诱变蛋白KatG(S315T)活性也有明显下降。荧光光谱显示KatG(R418Q)和KatG(A456S)的荧光强度较野生型明显降低。4个点突变均不影响KatG蛋白间二硫键的形成。结论 所引入的4个突变可能对KatG蛋白的结构造成影响,从而导致KatG蛋白的功能即过氧化氢-过氧化物酶活性下降。

Purpose: To study the effect of specical missense mutations on the structure and function of KatG from Mycobacterium tuberculosis. Methods: Site-directed mutagenesis was adopted to introduce specific mutation in the KatG gene. The mutant KatG genes were expressed in E. coli BL-21, and the catalase activity and peroxidase activity were determined. The steady-state tryptophan fluorescence spectra of the wild type and the mutant KatG proteins were measured. Results: Four mutant KatG genes were successfully obtained by site-mutagensis method. The mutant KatG genes were expressed and the catalase-peroxidase activities were determined. The disulphide-bridge formation of these recombinant proteins were analysed. The spectroscopic changes of three proteins were observed, too. It was found that each of the four special missense mutations (S315T, W321G, R418Q and A456S) caused a significant decrease in the catalase-peroxidase activity of KatG. The substitutions including S315T, W321G, R418Q and A456S did not affect the disulphide-bridge formation. The fluorescence intensity of KatG(R418Q) and KatG(A456S) decreased over 10% compared with that of KatG(wt) while KatG (S315T) had no apparent change. Conclusions: The results suggested that the four mutations may affect he structure and function of KatG by different mechanisms.