Dumas R, Job D, Ortholand J Y, Emeric G, Greiner A, Douce R
Unité Mixte C.N.R.S./Rhône-Poulenc (Unité associée au Centre National de la Recherche Scientifique, U.M. 41), Lyon, France.
Biochem J. 1992 Dec 15;288 ( Pt 3)(Pt 3):865-74. doi: 10.1042/bj2880865.
Acetohydroxy acid isomeroreductase catalyses a two-step reaction, an alkyl migration and a NADPH-dependent reduction, in the assembly of the carbon skeletons of branched-chain amino acids. Detailed investigations of acetohydroxy acid isomeroreductase aimed at elucidating the biosynthetic pathway of branched-chain amino acids and at designing new inhibitors of the enzyme having herbicidal potency have so far been conducted with the enzymes isolated from bacteria. To gain more information on a plant system, the gene encoding the mature acetohydroxy acid isomeroreductase from spinach (Spinacia oleracea) leaf chloroplasts has been used to transform Escherichia coli cells and to overexpress the enzyme. A rapid protocol is described that allows the preparation of large quantities of pure spinach chloroplast acetohydroxy acid isomeroreductase. Kinetic and structural properties of the plant enzyme expressed in Escherichia coli are compared with those reported in our previous studies on the native enzymes purified from spinach chloroplasts and with those reported for the corresponding enzymes isolated from Escherichia coli and Salmonella typhimurium. Both the plant and the bacterial enzymes obey an ordered mechanism in which NADPH binds first, followed by substrate (either 2-acetolactate or 2-aceto-2-hydroxybutyrate). Inhibition studies employing an inactive substrate analogue, 2-hydroxy-2-methyl-3-oxopentanoate, showed, however, that the binding of 2-hydroxy-2-methyl-3-oxopentanoate and NADPH occurs randomly, suggestive of some flexibility of the plant enzyme active site. The observed preference of the enzyme for 2-aceto-2-hydroxybutyrate over 2-acetolactate is discussed with regard to the contribution of acetohydroxy acid isomeroreductase activity in the partitioning between isoleucine and valine biosyntheses. Moreover, the kinetic properties of the chloroplast enzyme support the notion that biosynthesis of branched-chain amino acids in plants is controlled by light. As judged by analytical-ultracentrifugation and gel-filtration analyses the overexpressed plant enzyme is a dimer of identical subunits.
乙酰羟酸异构还原酶在支链氨基酸碳骨架的组装过程中催化一个两步反应，即烷基迁移和依赖于NADPH的还原反应。迄今为止，针对乙酰羟酸异构还原酶的详细研究旨在阐明支链氨基酸的生物合成途径，并设计具有除草效力的该酶新抑制剂，这些研究都是用从细菌中分离出的酶进行的。为了获取更多关于植物系统的信息，编码菠菜（Spinacia oleracea）叶叶绿体成熟乙酰羟酸异构还原酶的基因已被用于转化大肠杆菌细胞并使该酶过量表达。本文描述了一种快速方法，可用于制备大量纯的菠菜叶绿体乙酰羟酸异构还原酶。将在大肠杆菌中表达的植物酶的动力学和结构特性与我们之前对从菠菜叶绿体中纯化的天然酶的研究报告结果以及从大肠杆菌和鼠伤寒沙门氏菌中分离出的相应酶的研究报告结果进行了比较。植物酶和细菌酶均遵循有序机制，其中NADPH首先结合，随后是底物（2-乙酰乳酸或2-乙酰-2-羟基丁酸）。然而，使用无活性底物类似物2-羟基-2-甲基-3-氧代戊酸进行的抑制研究表明，2-羟基-2-甲基-3-氧代戊酸和NADPH的结合是随机发生的，这表明植物酶活性位点具有一定的灵活性。针对乙酰羟酸异构还原酶活性在异亮氨酸和缬氨酸生物合成分配中的作用，讨论了该酶对2-乙酰-2-羟基丁酸而非2-乙酰乳酸的观察到的偏好。此外，叶绿体酶的动力学特性支持了植物中支链氨基酸生物合成受光控制的观点。通过分析超速离心和凝胶过滤分析判断，过量表达的植物酶是由相同亚基组成的二聚体。
