Le Dung Tien, Yoon Moon-Young, Kim Young Tae, Choi Jung-Do
School of Life Sciences, Chungbuk National University, Cheongju 361-763, Korea.
J Biochem. 2005 Jul;138(1):35-40. doi: 10.1093/jb/mvi099.
Acetohydroxy acid synthase (AHAS, EC 2.2.1.6; also known as acetolactate synthase, ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in plants and microorganisms. AHAS is the target of several classes of herbicides. In the present study, the role of three well-conserved arginine residues (R141, R372, and R376) in tobacco AHAS was determined by site-directed mutagenesis. The mutated enzymes, referred to as R141A, R141F, and R376F, were inactive and unable to bind to the cofactor, FAD. The inactive mutants had the same secondary structure as that of the wild type. The mutants R141K, R372F, and R376K exhibited much lower specific activities than the wild type, and moderate resistance to herbicides such as Londax, Cadre, and/or TP. The mutation R141K showed a strong reduction in activation efficiency by ThDP, while the mutations R372K and R376K showed a strong reductions in activation efficiency by FAD in comparison to the wild type enzyme. Taking into account the data presented here and the homology model constructed previously [Le et al. (2004) Biochem. Biophys. Res. Commun. 317, 930-938], it is suggested that the three amino acid residues studied (R141, R372, and R376) are located essentially at the enzyme active site, and, furthermore, that residues R372 and R376 are possibly responsible for the binding of the enzyme to FAD.
乙酰羟酸合酶（AHAS，EC 2.2.1.6；也称为乙酰乳酸合酶，ALS）催化植物和微生物中缬氨酸、亮氨酸和异亮氨酸生物合成的首个共同步骤。AHAS是几类除草剂的作用靶点。在本研究中，通过定点诱变确定了烟草AHAS中三个保守的精氨酸残基（R141、R372和R376）的作用。突变酶，即R141A、R141F和R376F，没有活性且无法与辅因子FAD结合。无活性的突变体与野生型具有相同的二级结构。突变体R141K、R372F和R376K的比活性远低于野生型，并且对Londax、Cadre和/或TP等除草剂具有中等抗性。与野生型酶相比，R141K突变导致ThDP激活效率大幅降低，而R372K和R376K突变导致FAD激活效率大幅降低。考虑到本文提供的数据以及先前构建的同源模型[Le等人（2004年）《生物化学与生物物理研究通讯》317，930 - 938]，表明所研究的三个氨基酸残基（R141、R372和R376）基本位于酶活性位点，此外，R372和R376残基可能负责酶与FAD的结合。
