Structure and mechanism of action of isopentenylpyrophosphate-dimethylallylpyrophosphate isomerase
Structure and mechanism of action of isopentenylpyrophosphate-dimethylallylpyrophosphate isomerase
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DOI：
10.1021/ja029171p
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发表时间：
2003-03-19
影响因子：
15
通讯作者：
Oldfield, E
中科院分区：
文献类型：
--
作者：
Wouters, J;Oudjama, Y;Oldfield, E
我们获得了C67A突变型大肠杆菌异戊烯基焦磷酸二甲基烯基焦磷酸异构酶（EC 5.3.3.2）与异戊烯基焦磷酸溴丙烷配合的三维x射线晶体结构，分辨率为1.93 Å。在二价金属阳离子（Mn2+或Mg2+）存在的情况下，整个主链折叠与先前获得的野生型酶非常相似。然而，在新的结构中，有两个金属结合位点，而不仅仅是一个。第一个金属结合位点由Mn2+占据，与三个组氨酸和两个谷氨酸残基配位；第二个金属结合位点由Mg2+占据，与两个溴丙烷配体磷酸氧、A67的羰基氧、E87的羧基氧和两个水分子配位。溴丙烷抑制剂的C3羟基与两个mn结合的谷氨酸之一E116的羧基有一个短氢键。所得到的结构与酶的作用机制一致，在野生型酶中，E116的羧基使焦磷酸异戊烯基的双键质子化，形成一个碳正离子，然后被C67的硫酸盐去除一个C2质子。在溴丙烷抑制剂结构的基础上，也很容易解释多种其他有效抑制剂对酶的抑制作用。
We have obtained the three-dimensional X-ray crystallographic structure of a C67A mutantEscherichia coliisopentenylpyrophosphate-dimethylallylpyrophosphate isomerase (EC 5.3.3.2) complexed with the bromohydrin of isopentenylpyrophosphate, at 1.93 Å resolution. The overall backbone fold is very similar to that obtained previously for the wild-type enzyme in the presence of a divalent metal cation (Mn2+or Mg2+). However, in the new structure, there are two metal binding sites, not just one. The first metal binding site is occupied by Mn2+, coordinated to three histidine and two glutamate residues, while the second is occupied by Mg2+, coordinated to two bromohydrin-ligand phosphate oxygens, the carbonyl oxygen of A67, a carboxyl oxygen of E87, and two water molecules. The C3 hydroxyl group of the bromohydrin inhibitor is involved in a short hydrogen bond to the carboxyl group of E116, one of the two Mn-bound glutamates. The structure obtained is consistent with a mechanism of action of the enzyme in which the carboxyl group of E116 protonates the double bond in isopentenylpyrophosphate, forming a carbocation, followed by removal of a C2 proton by the thiolate of C67, in the wild-type enzyme. The inhibition of the enzyme by a wide variety of other potent inhibitors is also readily explained on the basis of the bromohydrin inhibitor structure.