除草剂靶酶—AHAS酶及基因突变体与除草剂设计(I).  野生型和突变型E. coli AHAS II 酶动力学性质的系统研究

Studies on Herbicide Design through Mutation on Herbicidal Target ——Acetohydroxyacid Synthase(I). Enzyme kinetics of wild type and mutants of E. coli AHAS II

摘要: 针对除草剂敏感型乙酰羟基酸合成酶E. coli AHAS II的抗性域,引入W464A、W464F、W464L、W464Y点突变。采用Megaprimer PCR定点突变,测序鉴定,构建了4个E. coli AHAS IIW464位点的突变体。通过对E. coli AHAS II野生型及突变体动力学性质的测定,发现它们对于底物—丙酮酸及3种辅助因子(FAD、ThDP、Mg2+)有着不同的特征常数。这些部分抗性酶系的建立以及对动力学性质的系统研究,为探讨AHAS酶对农药分子抗性的作用机制、设计合成新除草剂及其筛选体系提供了基础。

Abstract: To investigate the interaction of various mutation forms of acetohydroxyacid synthase (AHAS) with herbicides and study the mechanism of herbicide resistance, a series of W464 mutated AHAS isoenzyme II were constructed by megaprimer method. DNA sequencing showed that the four mutants of E. coli AHAS II are successfully constructed. These purified mutants have different properties on different enzyme cofactors, such as pyruvate, ThDP, FAD and Mg2+, compared to that of the wild type. The influences on Kc of FAD and Mg2+ by mutants showed the same trend: Wild-type > W464F > W464L > W464A >W464Y; while that of pyruvate and ThDP showed more dramatic changes. The systematic study on enzyme kinetics of wild type and mutant AHAS provides information for inhibition and resistant mechanism of herbicidal molecules on the target enzyme.