Fujiwara Kazuko, Hosaka Harumi, Matsuda Makoto, Okamura-Ikeda Kazuko, Motokawa Yutaro, Suzuki Mamoru, Nakagawa Atsushi, Taniguchi Hisaaki
Institute for Enzyme Research, the University of Tokushima, Tokushima 770-8503, Japan.
J Mol Biol. 2007 Aug 3;371(1):222-34. doi: 10.1016/j.jmb.2007.05.059. Epub 2007 May 26.
Lipoic acid is an essential cofactor of the alpha-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 A 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.
硫辛酸是α-酮酸脱氢酶复合体和甘氨酸裂解系统的必需辅因子。它共价连接到复合体亚基的特定赖氨酸残基上。牛硫辛酰胺转移酶（bLT）以硫辛酰-AMP为底物催化硫辛酸连接反应，形成硫辛酰化蛋白和AMP。为了在原子水平深入了解反应机制，我们测定了分辨率为2.10 Å的bLT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，硫辛酰-AMP结合在N端结构域的活性位点，呈U形构象。硫辛酰部分埋在疏水口袋中，形成范德华相互作用，而AMP部分在另一个隧道状腔中与bLT形成大量氢键。这些相互作用共同作用，将硫辛酰-AMP的C10原子暴露于bLT分子表面。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激发硫辛酰-AMP的C10原子的正电荷，从而促进硫辛酸受体蛋白的赖氨酸残基进行亲核攻击，同时伴随着羰基与磷酸基团之间的键断裂。我们讨论了bLT与来自大肠杆菌和嗜酸嗜热放线菌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也含有内源性硫辛酰单核苷酸，随时准备对脱辅基蛋白进行硫辛酰化。
