Nakai Tadashi, Nakagawa Noriko, Maoka Nobuko, Masui Ryoji, Kuramitsu Seiki, Kamiya Nobuo
RIKEN Harima Institute/SPring-8, 1-1-1 Kouto, Mikazuki, Sayo-gun, Hyogo 679-5148, Japan.
J Mol Biol. 2004 Apr 2;337(4):1011-33. doi: 10.1016/j.jmb.2004.02.011.
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 E1 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 E1 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.
支链2-氧代酸（BCOA）脱氢酶多酶复合物的α(2)β(2)四聚体E1组分是一种依赖硫胺二磷酸（ThDP）的酶。E1催化BCOA的脱羧反应，同时形成α-碳负离子/烯胺中间体2-(1-羟烷基)-ThDP，随后将1-羟烷基转移至E2组分脂酰胺远端的硫原子上。为阐明E1的催化机制，嗜热栖热菌HB8的E1的α亚基和β亚基已在大肠杆菌中共表达、纯化并结晶为稳定复合物，且已分析了以下晶体结构：脱辅酶（E1(apo)）、全酶（E1(holo)）、与底物类似物4-甲基戊酸（MPA）形成的ES复合物模型的E1(holo)以及与4-甲基-2-氧代戊酸（MOPA）形成α-碳负离子/烯胺中间体（E1(ceim)）的E1(holo)。辅因子与E1(apo)的结合在活性位点附近的两个环中诱导无序到有序的转变。此外，当MPA与E1(holo)结合时，由Gly121β-Gln131β组成的环靠近活性位点并与MPA相互作用。MPA的羧基主要由Tyr86β和ThDP的N4'识别。MPA的疏水部分由Phe66α、Tyr95α、Met128α和His131α识别。作为中间体，MOPA脱羧并与ThDP共价连接，且蛋白质环的构象与无底物（全酶）形式几乎相同。这些结果表明，E1在与BCOA形成ES复合物时经历开闭构象变化，且可移动区域参与对BCOA羧基的识别。由上述结构构建的E1(holo).MOPA和E1(ceim).脂酰胺的ES复合物模型表明，His273α和His129β'分别是BCOA羰基和脂酰胺近端硫原子的潜在质子供体。
