Crystal structure of bovine lipoyltransferase in complex with lipoyl-AMP.
Crystal structure of bovine lipoyltransferase in complex with lipoyl-AMP.
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DOI：
10.1016/j.jmb.2007.05.059
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发表时间：
2007-08
影响因子：
5.6
通讯作者：
K. Fujiwara;H. Hosaka;M. Matsuda;K. Okamura-Ikeda;Y. Motokawa;Mamoru Suzuki;A. Nakagawa;H. Taniguchi-H
中科院分区：
文献类型：
--
作者：
K. Fujiwara;H. Hosaka;M. Matsuda;K. Okamura-Ikeda;Y. Motokawa;Mamoru Suzuki;A. Nakagawa;H. Taniguchi-H
硫辛酸是 α-酮酸脱氢酶复合物和甘氨酸裂解系统的重要辅助因子。它共价连接到复合物亚基的特定赖氨酸残基上。牛硫辛酸转移酶（bLT）以硫辛酸-AMP为底物催化硫辛酸附着反应，形成硫辛酸化蛋白质和AMP。为了深入了解原子水平的反应机制，我们以 2.10 Å 分辨率确定了 bLT 的晶体结构。出乎意料的是，纯化的重组bLT含有内源性硫辛酰-AMP。 bLT的结构由N端和C端结构域组成，硫辛酸-AMP与N端结构域的活性位点结合，呈U形构象。硫辛酰部分埋在疏水袋中，形成范德华相互作用，AMP部分与另一个隧道状空腔中的bLT形成许多氢键。这些相互作用共同作用，将硫辛酰-AMP 的 C10 原子暴露到 bLT 分子的表面。硫辛酰-AMP 的羰基氧原子与不变的 Lys135 相互作用。这种相互作用可能会刺激硫辛酸-AMP C10 原子的正电荷，从而促进硫辛酸受体蛋白赖氨酸残基的亲核攻击，同时伴随着羰基和磷酸基之间的键断裂。我们讨论了 bLT 和来自大肠杆菌和嗜酸热原体的硫辛酸蛋白连接酶 A 之间的结构差异。我们进一步证明线粒体中的 bLT 还含有内源性脂酰单核苷酸，为脱辅基蛋白的脂酰化做好准备。
Lipoic acid is an essential cofactor of the α-ketoacid dehydrogenase complexes and the glycine cleavage system. It is covalently attached to a specific lysine residue of the subunit of the complexes. The bovine lipoyltransferase (bLT) catalyzes the lipoic acid attachment reaction using lipoyl-AMP as a substrate, forming a lipoylated protein and AMP. To gain insights into the reaction mechanism at the atomic level, we have determined the crystal structure of bLT at 2.10 Å resolution. Unexpectedly, the purified recombinant bLT contains endogenous lipoyl-AMP. The structure of bLT consists of N-terminal and C-terminal domains, and lipoyl-AMP is bound to the active site in the N-terminal domain, adopting a U-shaped conformation. The lipoyl moiety is buried in the hydrophobic pocket, forming van der Waals interactions, and the AMP moiety forms numerous hydrogen bonds with bLT in another tunnel-like cavity. These interactions work together to expose the C10 atom of lipoyl-AMP to the surface of the bLT molecule. The carbonyl oxygen atom of lipoyl-AMP interacts with the invariant Lys135. The interaction might stimulate the positive charge of the C10 atom of lipoyl-AMP, and consequently facilitate the nucleophilic attack by the lysine residue of the lipoate-acceptor protein, accompanying the bond cleavage between the carbonyl group and the phosphate group. We discuss the structural differences between bLT and the lipoate-protein ligase A from Escherichia coli and Thermoplasma acidophilum. We further demonstrate that bLT in mitochondria also contains endogenous lipoylmononucleotide, being ready for the lipoylation of apoproteins.