Ligand-induced conformational changes and a reaction intermediate in branched-chain 2-oxo acid dehydrogenase (E1) from Thermus thermophilus HB8, as revealed by X-ray crystallography
Ligand-induced conformational changes and a reaction intermediate in branched-chain 2-oxo acid dehydrogenase (E1) from Thermus thermophilus HB8, as revealed by X-ray crystallography
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DOI：
10.1016/j.jmb.2004.02.011
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发表时间：
2004-04-02
影响因子：
5.6
通讯作者：
Kamiya, N
中科院分区：
文献类型：
--
作者：
Nakai, T;Nakagawa, N;Kamiya, N
支链2-含氧酸（BCOA）脱氢酶多酶复合物的α（2）β（2）四聚体E1组分是一种二磷酸硫胺素（ThDP）依赖性酶。E1催化BCOA的脱羧，同时形成α-碳负离子/烯胺中间体2-（1-羟基烷基）-ThDP，然后将1-羟基烷基转移到E2组分的硫辛酰胺上的远端硫原子上。为了阐明E1的催化机制，来自嗜热栖热菌HB 8的E1的α-和β-亚基已经在大肠杆菌中共表达，纯化并结晶为稳定的复合物，并且已经分析了以下晶体结构：脱辅基酶（E1（apo）），全酶（E1（holo）），E1（holo）与底物类似物4-甲基戊酸（MPA）复合作为ES复合物模型，和E1（holo）与作为α-碳负离子/烯胺中间体的4-甲基-2-氧代戊酸酯（MOPA）复合（E1（ceim））。辅因子与E1（载脂蛋白）的结合在活性位点附近的两个环中诱导无序-有序转变。此外，在MPA与E1（holo）结合后，由Gly 121 β-Gln 131 β组成的环移动靠近活性位点并与MPA相互作用。MPA的羧酸基团主要被ThDP的Tyr 86 β和N4'识别。MPA的疏水部分被Phe 66 α、Tyr 95 α、Met 128 α和His 131 α识别。作为中间体，MOPA脱羧并共价连接到ThDP，蛋白质环的构象几乎与无底物（全酶）形式相同。这些结果表明，E1与BCOA形成ES复合物时经历了开-闭构象变化，移动的区域参与了BCOA羧基的识别. E1（holo）（.）MOPA和E1（ceim）（.）由上述结构构建的硫辛酰胺表明，His 273 α和His 129 β ′分别是BCOA的羰基和硫辛酰胺上邻近硫原子的潜在质子供体。(C)2004 Elsevier Ltd.保留所有权利。
The alpha(2)beta(2) tetrameric E1 component of the branched-chain 2-oxo acid (BCOA) dehydrogenase multienzyme complex is a thiamin diphosphate (ThDP)-dependent enzyme. E1 catalyzes the decarboxylation of a BCOA concomitant with the formation of the alpha-carbanion/enamine intermediate, 2-(1-hydroxyalkyl)-ThDP, followed by transfer of the 1-hydroxyalkyl group to the distal sulfur atom on the lipoamide of the E2 component. In order to elucidate the catalytic mechanism of E1, the alpha- and beta-subunits of El from Thermus thermophilus HB8 have been co-expressed in Escherichia coli, purified and crystallized as a stable complex, and the following crystal structures have been analyzed: the apoenzyme (E1(apo)), the holoenzyme (E1(holo)), E1(holo) in complex with the substrate analogue 4-methylpentanoate (MPA) as an ES complex model, and E1(holo) in complex with 4-methyl-2-oxopentanoate (MOPA) as the alpha-carbanion/enamine intermediate (E1(ceim)). Binding of cofactors to E1(apo) induces a disorder-order transition in two loops adjacent to the active site. Furthermore, upon binding of MPA to E1(holo), the loop comprised of Gly121beta-Gln131beta moves close to the active site and interacts with MPA. The carboxylate group of MPA is recognized mainly by Tyr86beta and N4' of ThDP. The hydrophobic moiety of MPA is recognized by Phe66alpha, Tyr95alpha, Met128alpha and His131alpha. As an intermediate, MOPA is decarboxylated and covalently linked to ThDP, and the conformation of the protein loop is almost the same as in the substrate-free (holoenzyme) form. These results suggest that El undergoes an open-closed conformational change upon formation of the ES complex with a BCOA, and the mobile region participates in the recognition of the carboxylate group of the BCOA. ES complex models of E1(holo)(.)MOPA and of E1(ceim)(.)lipoamide built from the above structures suggest that His273alpha and His129beta' are potential proton donors to the carbonyl group of a BCOA and to the proximal sulfur atom on the lipoamide, respectively. (C) 2004 Elsevier Ltd. All rights reserved.