Characterization of Biotin Transporters

生物素是一种维生素和辅因子，在脂肪酸生物合成等生物过程中发挥着重要作用。生物素要么在细胞内合成，要么通过质膜运输，然后附着在生物素依赖酶的特定赖氨酸残基上。因此，这些过程中的一个或全部出现故障，以及需要生物素的蛋白质出现缺陷，都会导致生物体出现严重的紊乱。为了更详细地阐明生物素在细胞过程中的作用，本项目进行了全面的研究，特别强调了生物素在细胞膜上的转运。生物素转运蛋白在大肠杆菌和酵母菌中已经得到了广泛的研究，但它们的化学实体没有被识别出来。根据已知的大肠杆菌转运蛋白基因(Biop)位于遗传图谱上的第86分钟，搜索数据库以寻找可能的候选者(S)。发现o461基因符合所有的…。对(生物素)转运蛋白的要求更高；推导出的氨基酸序列表明它是一种疏水蛋白质，并与已知的来自不同来源的氨基酸转运蛋白具有相当大的同源性。该基因的缺失导致了大肠杆菌生物素转运活性的显著下降。除了生物素在大肠杆菌中的转运外，还用乙酰辅酶A羧化酶(ACC)和丙酮酸羧化酶(PC)研究了生物素的催化作用。大肠杆菌ACC由三个组分组成：生物素羧基酶(BC)、羧基转移酶和生物素羧基载体蛋白(BCCP)，而嗜热脂肪芽孢杆菌的PC是单一的多肽链。采用了两种方法：(L)对可能的活性中心残基进行定点突变；(2)构建Acc和PC嵌合酶。在后者中，PC的功能结构域BC和BCCP被相应的ACC结构域取代。对这些嵌合体的初步研究提供了关于生物素如何与羧基酶的各个区域相互作用的信息。较少

Biotin is a vitamin and cofactor and plays an important role in such biological processes as fatty acid biosynthesis. Biotin is either synthesized within the cells or transported across plasma membranes and then attached to a specific lysine residue of biotin-dependent enzymes. Hence, malfunctioning of either one or all of these processes as well as defects in biotin-requiring proteins result in severe disorders in organisms. In order to clarify the function of biotin in cellular processes in more detail, comprehensive studies were undertaken in this project with special emphasis on biotin transport across cell membranes. Biotin transporters have been studied extensively in Escherichia coli and yeast, Saccharomyces cerevisiae, but their chemical entities escaped identification. On the basis of previous knowledge on the E. coli transporter that its gene (bioP) resides at 86 min on the genetic map, data base was searched for a probable candidate(s). It was found that gene o461 meets all … More the requirements for a (biotin) transporter; the deduced amino acid sequence suggests that it is a hydrophobic protein and shows considerable homology with those of known amino acid transporters from various sources. Disruption of this gene resulted in a significant decrease in the biotin transport activity of E. coli. In addition to the biotin transport in E. coli, the catalytic function of biotin was studied with acetyl-CoA carboxylase (ACC) and pyruvate carboxylase (PC). E. coli ACC is made of three components: biotin carboxylase (BC), carboxyl transferase and biotin carboxyl carrier protein (BCCP), while PC from Bacillus stearothermophilus is of a single polypeptide chain. Two approaches were taken, (l) site-directed mutagenesis of putative active site residues and (2) construction of chimeric enzymes of ACC and PC. In the latter, functional domains BC and BCCP of PC were replaced with the corresponding domains of ACC. Preliminary studies of these chimeras presented information on how biotin interacts with the individual domains of the carboxylases. Less