Li S J, Cronan J E
Department of Microbiology, University of Illinois, Urbana 61801.
J Biol Chem. 1992 Jan 15;267(2):855-63.
We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the alpha-subunit of rat propionyl-CoA carboxylase. The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role. The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed. As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass. However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein. Amino-terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties. Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene. We have also determined the mutational alteration in a previously isolated acetyl-CoA carboxylase (fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin. Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to beta-galactosidase resulted in biotinated beta-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.
我们报道了编码大肠杆菌乙酰辅酶A羧化酶生物素羧化酶亚基的基因的分子克隆及DNA序列。生物素羧化酶基因编码一个由449个残基组成的蛋白质，该蛋白质与两种生物素依赖性羧化酶蛋白（酵母丙酮酸羧化酶和大鼠丙酰辅酶A羧化酶的α亚基）的氨基末端片段惊人地相似。推导的生物素羧化酶序列包含一个共有ATP结合位点以及在所有已测序的依赖碳酸氢盐的生物素羧化酶中都保守的含半胱氨酸序列，该序列可能起关键催化作用。编码乙酰辅酶A羧化酶生物素羧基载体蛋白（BCCP）亚基的基因位于生物素羧化酶基因的上游，且这两个基因共同转录。正如其他人之前所报道的，BCCP序列编码一个分子量为16,688的蛋白质。然而，这个值比通过蛋白质分析得到的值（22,500道尔顿）小得多。纯化的BCCP蛋白的氨基末端氨基酸测序证实了推导的氨基酸序列，表明BCCP是一种具有非典型物理性质的蛋白质。Northern印迹分析和引物延伸分析表明，BCCP和生物素羧化酶作为单一的mRNA物种转录，该mRNA在BCCP基因之前包含一个异常长的非翻译前导序列。我们还确定了先前分离的乙酰辅酶A羧化酶（fabE）突变体中的突变改变，并表明该损伤位于BCCP基因内，导致产生一种在接受生物素方面有缺陷的BCCP物种。BCCP羧基末端110个或84个（但不是76个）氨基酸与β-半乳糖苷酶的翻译融合产生了生物素化的β-半乳糖苷酶分子，并且已证明产生一种这样的融合会导致生物素生物合成操纵子的去阻遏。
