Jung Sun-Mi, Le Dung Tien, Yoon Sung-Sook, Yoon Moon-Young, Kim Young Tae, Choi Jung-Do
School of Life Sciences and Biotechnology Research Institute, Chungbuk National University, Cheongju 361-763, Korea.
Biochem J. 2004 Oct 1;383(Pt 1):53-61. doi: 10.1042/BJ20040720.
The enzyme AHAS (acetohydroxy acid synthase), which is involved in the biosynthesis of valine, leucine and isoleucine, is the target of several classes of herbicides. A model of tobacco AHAS was generated based on the X-ray structure of yeast AHAS. Well conserved residues at the herbicide-binding site were identified, and the roles of three of these residues (Phe-205, Val-570 and Phe-577) were determined by site-directed mutagenesis. The Phe-205 mutants F205A, F205H, F205W and F205Y showed markedly decreased levels of catalytic efficiency, and cross-resistance to two or three classes of herbicides, i.e. Londax (a sulphonylurea herbicide), Cadre (an imidazolinone herbicide) and TP (a triazolopyrimidine derivative). None of the mutations caused significant changes in the secondary or tertiary structure of the enzyme. Four mutants of Phe-577, i.e. F577D, F577E, F577K and F577R, showed unaltered V(max) values, but substantially decreased catalytic efficiency. However, these mutants were highly resistant to two or three of the tested herbicides. The three mutants F577D, F577E and F577R had a similar secondary structure to that of wild-type AHAS. Conservative mutations of Phe-577, i.e. F577W and F577Y, did not affect the kinetic properties of the enzyme or its inhibition by herbicides. The mutation Val-570 to Asn abolished the binding affinity of the enzyme for FAD as well as its activity, and also caused a change in the tertiary structure of AHAS. However, the mutant V570Q was active, but resistant to two classes of herbicides, i.e. Londax and TP. The conservative mutant V570I was substantially reduced in catalytic efficiency and moderately resistant to the three herbicides. The results of this study suggest that residues Phe-205, Val-570 and Phe-577 in tobacco AHAS are located at or near the binding site that is common for the three classes of herbicides. In addition, Phe-205 and Val-570 are probably located at the herbicide-binding site that may overlap partially with the active site. Selected mutants of Phe-577 are expected to be utilized to construct herbicide-resistant transgenic plants.
参与缬氨酸、亮氨酸和异亮氨酸生物合成的酶AHAS（乙酰羟酸合酶）是几类除草剂的作用靶点。基于酵母AHAS的X射线结构构建了烟草AHAS模型。确定了除草剂结合位点上保守性良好的残基，并通过定点诱变确定了其中三个残基（苯丙氨酸-205、缬氨酸-570和苯丙氨酸-577）的作用。苯丙氨酸-205突变体F205A、F205H、F205W和F205Y的催化效率水平显著降低，并对两类或三类除草剂产生交叉抗性，即Londax（一种磺酰脲类除草剂）、Cadre（一种咪唑啉酮类除草剂）和TP（一种三唑并嘧啶衍生物）。这些突变均未导致该酶二级或三级结构发生显著变化。苯丙氨酸-577的四个突变体，即F577D、F577E、F577K和F577R，其V(max)值未改变，但催化效率大幅降低。然而，这些突变体对两种或三种受试除草剂具有高度抗性。三个突变体F577D、F577E和F577R具有与野生型AHAS相似的二级结构。苯丙氨酸-577的保守突变，即F577W和F577Y，不影响该酶的动力学性质或其对除草剂的抑制作用。缬氨酸-570突变为天冬酰胺消除了该酶对FAD的结合亲和力及其活性，还导致了AHAS三级结构的变化。然而，突变体V570Q具有活性，但对两类除草剂，即Londax和TP具有抗性。保守突变体V570I的催化效率大幅降低，对三种除草剂具有中度抗性。本研究结果表明，烟草AHAS中的残基苯丙氨酸-205、缬氨酸-570和苯丙氨酸-577位于三类除草剂的共同结合位点处或附近。此外，苯丙氨酸-205和缬氨酸-570可能位于除草剂结合位点，该位点可能与活性位点部分重叠。预计苯丙氨酸-577的选定突变体将用于构建抗除草剂转基因植物。
