The crystal structure of plant acetohydroxy acid isomeroreductase complexed with NADPH, two magnesium ions and a herbicidal transition state analog determined at 1.65 angstrom resolution
The crystal structure of plant acetohydroxy acid isomeroreductase complexed with NADPH, two magnesium ions and a herbicidal transition state analog determined at 1.65 angstrom resolution
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DOI：
10.1093/emboj/16.12.3405
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发表时间：
1997-06-16
期刊：
影响因子：
11.4
通讯作者：
PebayPeyroula, E
中科院分区：
文献类型：
--
作者：
Biou, V;Dumas, R;PebayPeyroula, E
乙酰羟酸异构还原酶催化乙酰羟酸转化为二羟基戊酸。该反应是必需的支链氨基酸缬氨酸和异亮氨酸合成途径中的第二个反应。由于这种途径在动物中不存在，因此参与其中的酶是系统搜索除草剂的良好目标。与辅因子NADPH、Mg 2+离子和具有除草活性的竞争性抑制剂N-羟基-N-异丙基草胺酸盐络合的乙酰羟酸异构还原酶的晶体结构被解析至1.65埃分辨率，并被精修至18.7%的R因子和22.9%的R游离。不对称单元显示了两个由非晶体学对称性相关的功能二聚体。活性位点，嵌套在界面之间的NADPH结合结构域和全螺旋的C-末端结构域，显示出类似的过渡态的情况。它含有两个Mg 2+离子与抑制剂分子相互作用，并通过天冬氨酸残基的羧酸根部分桥接。它的疏水口袋和极性区域的结合位点是很好的调整。在已知的乙酰羟酸异构还原酶序列中，只有24个氨基酸是保守的，并且所有这些氨基酸都位于活性位点周围。最后，一个140个氨基酸的区域，存在于植物中，但不存在于其他物种，被发现构成大部分的二聚化结构域。
Acetohydroxy acid isomeroreductase catalyzes the conversion of acetohydroxy acids into dihydroxy valerates. This reaction is the second in the synthetic pathway of the essential branched side chain amino acids valine and isoleucine. Because this pathway is absent from animals, the enzymes involved in it are good targets for a systematic search for herbicides. The crystal structure of acetohydroxy acid isomeroreductase complexed with cofactor NADPH, Mg2+ ions and a competitive inhibitor with herbicidal activity, N-hydroxy-N-isopropyloxamate, was solved to 1.65 Angstrom resolution and refined to an R factor of 18.7% and an R free of 22.9%. The asymmetric unit shows two functional dimers related by non-crystallographic symmetry. The active site, nested at the interface between the NADPH-binding domain and the all-helical C-terminus domain, shows a situation analogous to the transition state. It contains two Mg2+ ions interacting with the inhibitor molecule and bridged by the carboxylate moiety of an aspartate residue. The inhibitor-binding site is well adjusted to it, with a hydrophobic pocket and a polar region. Only 24 amino acids are conserved among known acetohydroxy acid isomeroreductase sequences and all of these are located around the active site. Finally, a 140 amino acid region, present in plants but absent from other species, was found to make up most of the dimerization domain.