Molecular-biological analysis of eu34mps imolued in biosym thes is and tromfer of lipoid acid in plants.

硫辛酸是一种辅酶，对丙酮酸脱氢酶和甘氨酸脱羧酶等酶复合物的活性至关重要。尽管在参与中枢代谢的酶复合物的功能中，硫辛酸假体具有重要的作用，但在任何生物体中，硫辛酸的生物合成和向同源蛋白的转移尚未得到很好的理解。在本研究中，我分离并鉴定了拟南芥中硫辛酸合成酶和脂酰转移酶的LIP1和LIP2 cdna。LIP1 cDNA编码拟南芥硫辛酸合成酶，通过其补充硫辛酸合成酶缺陷的大肠杆菌突变体的能力。LIP1 cDNA的DNA序列分析显示一个开放阅读框架，预测一个374个氨基酸的蛋白质。南方杂交分析表明，LIP1基因在拟南芥中为单拷贝基因。硫辛酸合成酶抗体的Western blot分析表明，硫辛酸合成酶以43 KD多肽的形式存在于拟南芥细胞的线粒体室中。在拟南芥中也克隆到了一个编码脂基转移酶的LIP2 cDNA。该cDNA编码脂酰基转移酶的能力证明它能够补充大肠杆菌脂酰基转移酶缺陷突变体（Lpla和LipB）。LIP2 cDNA的DNA序列分析显示了一个开放阅读框架，预测了235个氨基酸的蛋白质。南方和北方杂交分析表明，LIp2是一个单拷贝基因，在拟南芥叶片中以860个核苷酸的mRNA表达。脂酰基转移酶抗体的western blot分析表明，脂酰基转移酶位于拟南芥细胞的线粒体室中，是一个29 kDa的多肽。

Lipoic acid is a coenzyme that is essential for activity of enzyme complexes such as the pyruvate dehydrogenase and glycine decarboxylase complexes. Despite the importance of the lipoyl prosthetic group in the function of the enzyme complexes involved in central metabolism, the biosynthesis and transter of lipoic acid to cognate proteins have not been well understood in any organism.In this study I have isolated and characterized LIP1 and LIP2 cDNAs for lipoic acid synthase and lipoyltransferase, respectively, of Arabidopsis thaliana. The LIP1 cDNA was shown to code for Arabidopsis lipoic acid synthase by its ability to complement an E. coli mutant that is defective in lipoic acid synthase. DNA sequence analysis of the LIP1 cDNA revealed an open-reading frame predicting a protein of 374 amino acids. Southern hybridization analysis suggests that LIP1 is a single0copy gene in Arabidopsis. Western blot analysis with an antibody against lipoic acid synthase demonstrates that lipoic acid synthase is located in the mitochondrial compartment in Arabidopsis cells as a 43 KD polypeptide. A LIP2 cDNA encoding lipoyltransferase has also been cloned from Arabidopdis. This cDNA was shown to code for lipoyltransterase by its ability to complement a mutant of E. coli defective in lipoyltransferases (Lpla and LipB). DNA sequence analysis of the LIP2 cDNA revealed an open-reading frame predicting a protein of 235 amino acids. Southern and northern hybridization analyses suggest that LIp2 is a single-copy gene and expressed as a mRNA of 860 rucleotides in Arabidopsis leaves. Westem blot analysis with an antibody against lipoyltransferase demonstrates that lipoyltransferase is located in the mitochondrial compartment in Arabidopsis cells as a 29 kDa polypeptide.