Crystal structure of lipoate-protein ligase A bound with the activated intermediate - Insights into interaction with lipoyl domains
Crystal structure of lipoate-protein ligase A bound with the activated intermediate - Insights into interaction with lipoyl domains
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DOI：
10.1074/jbc.m507284200
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发表时间：
2005-11-11
影响因子：
4.8
通讯作者：
Suh, SW
中科院分区：
文献类型：
--
作者：
Kim, DJ;Kim, KH;Suh, SW
硫辛酸是催化关键代谢反应的几种多组分酶复合物的共价辅助因子。硫辛酸与脂酰依赖酶的结合是由脂酰-蛋白连接酶（LPLs）催化的。在大肠杆菌中，两种不同的酶脂质蛋白连接酶A （LplA）和脂质编码脂基转移酶（LipB）催化目标蛋白脂酰化的独立途径。LplA催化的反应分两步进行。首先，LplA以ATP为代价激活外源供应的硫辛酸，使其变成脂酰amp。接下来，它将酶结合的脂酰amp转移到脂酰结构域特定赖氨酸残基的ε -氨基上，从而形成酰胺键。为了深入了解LplA的作用机制，我们确定了嗜酸热原体LplA的三种形式的晶体结构：(i)载脂蛋白形式；（ii） ATP复合物；（iii）脂酰- amp复合物。LplA的整体折叠与大肠杆菌生物素全酶合成酶/生物抑制因子（BirA）的生物素基蛋白连接酶模块有一定的相似性。Lipoyl-AMP深深束缚在LplA的分叉口袋中，呈u形构象。脂酰amp只有磷酸基团和部分核糖可以通过隧道状通道从散装溶剂中进入，而其余的活化中间体则完全埋在活性位点口袋中。这种结合LplA的活化中间体的第一种观点使我们能够提出Ta LplA和脂酰结构域之间复合物的模型，从而阐明LplA的靶蛋白/赖氨酸残基特异性。
Lipoic acid is the covalently attached cofactor of several multicomponent enzyme complexes that catalyze key metabolic reactions. Attachment of lipoic acid to the lipoyl-dependent enzymes is catalyzed by lipoate-protein ligases (LPLs). In Escherichia coli, two distinct enzymes lipoate-protein ligase A (LplA) and lipB-encoded lipoyltransferase ( LipB) catalyze independent pathways for lipoylation of the target proteins. The reaction catalyzed by LplA occurs in two steps. First, LplA activates exogenously supplied lipoic acid at the expense of ATP to lipoyl-AMP. Next, it transfers the enzyme-bound lipoyl-AMP to the epsilon-amino group of a specific lysine residue of the lipoyl domain to give an amide linkage. To gain insight into the mechanism of action by LplA, we have determined the crystal structure of Thermoplasma acidophilum LplA in three forms: (i) the apo form; (ii) the ATP complex; and (iii) the lipoyl-AMP complex. The overall fold of LplA bears some resemblance to that of the biotinyl protein ligase module of the E. coli biotin holoenzyme synthetase/ bio repressor (BirA). Lipoyl-AMP is bound deeply in the bifurcated pocket of LplA and adopts a U-shaped conformation. Only the phosphate group and part of the ribose sugar of lipoyl-AMP are accessible from the bulk solvent through a tunnel-like passage, whereas the rest of the activated intermediate is completely buried inside the active site pocket. This first view of the activated intermediate bound to LplA allowed us to propose a model of the complexes between Ta LplA and lipoyl domains, thus shedding light on the target protein/lysine residue specificity of LplA.