Bar-Ilan A, Balan V, Tittmann K, Golbik R, Vyazmensky M, Hübner G, Barak Z, Chipman D M
Department of Life Sciences, Ben-Gurion University of the Negev, P.O.B. 653, Beer-Sheva 84105, Israel.
Biochemistry. 2001 Oct 2;40(39):11946-54. doi: 10.1021/bi0104524.
Acetohydroxyacid synthases (AHASs) are biosynthetic thiamin diphosphate- (ThDP) and FAD-dependent enzymes. They are homologous to pyruvate oxidase and other members of a family of ThDP-dependent enzymes which catalyze reactions in which the first step is decarboxylation of a 2-ketoacid. AHAS catalyzes the condensation of the 2-carbon moiety, derived from the decarboxylation of pyruvate, with a second 2-ketoacid, to form acetolactate or acetohydroxybutyrate. A structural model for AHAS isozyme II (AHAS II) from Escherichia coli has been constructed on the basis of its homology with pyruvate oxidase from Lactobacillus plantarum (LpPOX). We describe here experiments which further test the model, and test whether the binding and activation of ThDP in AHAS involve the same structural elements and mechanism identified for homologous enzymes. Interaction of a conserved glutamate with the N1' of the ThDP aminopyrimidine moiety is involved in activation of the cofactor for proton exchange in several ThDP-dependent enzymes. In accord with this, the analogue N3'-pyridyl thiamin diphosphate does not support AHAS activity. Mutagenesis of Glu47, the putative conserved glutamate, decreases the rate of proton exchange at C-2 of bound ThDP by nearly 2 orders of magnitude and decreases the turnover rate for the mutants by about 10-fold. Mutant E47A also has altered substrate specificity, pH dependence, and other changes in properties. Mutagenesis of Asp428, presumed on the basis of the model to be the crucial carboxylate ligand to Mg(2+) in the "ThDP motif", leads to a decrease in the affinity of AHAS II for Mg(2+). While mutant D428N shows ThDP affinity close to that of the wild-type on saturation with Mg(2+), D428E has a decreased affinity for ThDP. These mutations also lead to dependence of the enzyme on K(+). These experiments demonstrate that AHAS binds and activates ThDP in the same way as do pyruvate decarboxylase, transketolase, and other ThDP-dependent enzymes. The biosynthetic activity of AHAS also involves many other factors beyond the binding and deprotonation of ThDP; changes in the ligands to ThDP can have interesting and unexpected effects on the reaction.
乙酰羟酸合酶（AHASs）是生物合成的硫胺素二磷酸（ThDP）和FAD依赖性酶。它们与丙酮酸氧化酶以及ThDP依赖性酶家族的其他成员同源，这些酶催化的反应第一步是2-酮酸的脱羧反应。AHAS催化由丙酮酸脱羧衍生而来的2-碳部分与另一种2-酮酸缩合，形成乙酰乳酸或乙酰羟丁酸。基于与植物乳杆菌丙酮酸氧化酶（LpPOX）的同源性，构建了大肠杆菌AHAS同工酶II（AHAS II）的结构模型。我们在此描述了进一步测试该模型的实验，并测试了AHAS中ThDP的结合和激活是否涉及与同源酶相同的结构元件和机制。在几种ThDP依赖性酶中，保守的谷氨酸与ThDP氨基嘧啶部分的N1'相互作用参与了辅因子质子交换的激活。与此一致，类似物N3'-吡啶基硫胺素二磷酸不支持AHAS活性。假定的保守谷氨酸Glu47的诱变使结合的ThDP的C-2处的质子交换速率降低了近2个数量级，并使突变体的周转速率降低了约10倍。突变体E47A还改变了底物特异性、pH依赖性以及其他性质变化。基于模型推测，Asp428是“ThDP基序”中与Mg(2+)关键的羧酸盐配体，对其进行诱变导致AHAS II对Mg(2+)的亲和力降低。虽然突变体D428N在Mg(2+)饱和时显示出与野生型接近的ThDP亲和力，但D428E对ThDP的亲和力降低。这些突变还导致酶对K(+)的依赖性。这些实验表明，AHAS与丙酮酸脱羧酶、转酮醇酶和其他ThDP依赖性酶一样，以相同的方式结合和激活ThDP。AHAS的生物合成活性还涉及ThDP结合和去质子化之外的许多其他因素；ThDP配体的变化可能对反应产生有趣且意想不到的影响。
