Identification of the gene encoding lipoate-protein ligase A of Escherichia coli. Molecular cloning and characterization of the lplA gene and gene product.
Identification of the gene encoding lipoate-protein ligase A of Escherichia coli. Molecular cloning and characterization of the lplA gene and gene product.
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发表时间：
1994-06
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通讯作者：
T. Morris;Kelynne E. Reed;J. J. Cronan-J.
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作者：
T. Morris;Kelynne E. Reed;J. J. Cronan-J.
R(+)-硫辛酸是α-酮酸脱氢酶和甘氨酸裂解酶复合体发挥功能所必需的辅因子。天然形式的脂酸盐通过酰胺键连接到保守的接受脂类的蛋白质结构域中特定赖氨酸残基的epsilon-氨基上。脂酸盐-蛋白连接酶(S)催化硫辛基和特定脱脂蛋白之间的这种酰胺键的形成。我们报道了编码大肠杆菌脂蛋白连接酶的lplA基因的分离。LplA缺失突变的菌株正常转运硫辛酸，但在结合和利用外源供应的硫辛酸和硫辛酸类似物方面存在严重缺陷。这些菌株对亚硒酸盐(一种抑制生长的脂酸盐类似物)也具有高度的抗性，并且在细胞提取物中不含有可检测到的脂酸盐蛋白连接酶活性。LplA基因已经被克隆、测序，并被物理定位到大肠杆菌染色体的最小99.6(4657千碱基)上。过表达后，38 kDa的lplA基因产物被纯化为均一的产物，其质量、N端序列和氨基酸组成与推导出的337个残基的一级序列一致。酶分析表明，纯化的LplA催化依赖于ATP的[35S]硫辛酸与载脂蛋白的结合，从而证实了LplA编码脂酸盐-蛋白连接酶A。对LplA缺失突变株的分析还表明，在大肠杆菌中存在第二个不依赖于LplA的硫酰连接酶。这是首次鉴定的脂酸盐连接酶基因，也是首次对纯化的脂酸盐连接酶进行分析。
R(+)-Lipoic acid is a cofactor required for function of the alpha-keto acid dehydrogenase and glycine cleavage enzyme complexes. The naturally occurring form of lipoate is attached by amide linkage to the epsilon-amino group of a specific lysine residue within conserved lipoate-accepting protein domains. Lipoate-protein ligase(s) catalyze the formation of this amide bond between lipoyl groups and specific apoproteins. We report the isolation of the lplA gene which encodes an Escherichia coli lipoate-protein ligase. Strains with lplA null mutations transport lipoic acid normally but have severe defects in the incorporation and utilization of exogenously supplied lipoic acid and lipoic acid analogs. These strains are also highly resistant to selenolipoate (a growth-inhibiting lipoate analog) and contain no detectable lipoate-protein ligase activity in cell extracts. The lplA gene has been cloned, sequenced, and physically mapped to min 99.6 (4657 kilobases) of the E. coli chromosome. Upon overexpression, the 38-kDa lplA gene product was purified to homogeneity and shown to have a mass, N-terminal sequence and amino acid composition consistent with the deduced 337 residue primary sequence. Enzyme assays show that purified LplA catalyzes the ATP-dependent attachment of [35S]lipoic acid to apoprotein, thus confirming that lplA encodes lipoate-protein ligase A. Analysis of lplA null mutants also indicates the existence of a second (lplA-independent) lipoyl-ligase enzyme in E. coli. This is the first identification of a lipoate ligase gene and the first analysis of a purified lipoate ligase enzyme.