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体的活性是必不可少的。尽管硫辛基修复基在参与中枢代谢的酶复合体的功能中起着重要的作用，但硫辛酸的生物合成和转运在任何生物体中都没有被很好地理解。在本研究中，我分离并鉴定了拟南芥硫辛酸合成酶和硫辛基转移酶的LIP1和LIP2 cDNA。LIP1基因编码了拟南芥硫辛酸合成酶，因为它能够补充硫辛酸合成酶缺陷的大肠杆菌突变体。对LIP1基因的DNA序列分析显示，LIP1有一个开放阅读框，预测了一个由374个氨基酸组成的蛋白质。Southern杂交分析表明，LIP1基因在拟南芥中是一个单拷贝基因。用硫辛酸合成酶抗体进行Western印迹分析表明，硫辛酸合成酶以43kD的多肽形式定位于拟南芥细胞的线粒体内。从拟南芥中还克隆到了一个编码硫辛基转移酶的LIP2基因。该基因编码硫酰基转移酶，因为它能补充硫酰基转移酶缺陷的大肠杆菌突变株(LplA和LipB)。对LIP2基因的DNA序列分析显示，该基因有一个开放阅读框，预测了一个235个氨基酸的蛋白质。Southern和Northern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形式表达。用抗硫转移酶的抗体进行免疫印迹分析表明，硫转移酶以29 kDa的多肽形式定位于拟南芥细胞的线粒体内。

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.