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起着重要作用。生物素要么在细胞内合成，要么穿过质膜运输，然后附着在生物素依赖性酶的特定赖氨酸残基上。因此，一个或所有这些过程的故障以及生物素需要蛋白质的缺陷会导致生物体的严重疾病。为了更详细地阐明生物素在细胞过程中的功能，本项目进行了全面的研究，特别强调生物素跨细胞膜的转运。生物素转运蛋白在大肠杆菌、酵母、酿酒酵母中已被广泛研究，但其化学实体未能得到鉴定。在前人对E. coli转运蛋白基因bioP在遗传图谱上位于86 min处，在数据库中搜索可能的候选者。发现o 461基因符合所有 ...更多信息 对（生物素）转运蛋白的要求;推导的氨基酸序列表明，它是一种疏水蛋白，并与来自各种来源的已知氨基酸转运蛋白显示出相当大的同源性。该基因的破坏导致E.杆菌除了E.在大肠杆菌中，研究了乙酰辅酶A羧化酶（ACC）和丙酮酸羧化酶（PC）对生物素的催化作用。E.大肠杆菌ACC由生物素羧化酶（BC）、羧基转移酶和生物素羧基载体蛋白（BCCP）三部分组成，而嗜热脂肪芽孢杆菌PC为单链多肽。采用两种方法，（1）对推定的活性位点残基进行定点突变和（2）构建ACC和PC的嵌合酶。在后者中，PC的功能结构域BC和BCCP被替换为ACC的相应结构域。这些嵌合体的初步研究提供了关于生物素如何与羧化酶的各个结构域相互作用的信息。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