Cronan J E, Li W B, Coleman R, Narasimhan M, de Mendoza D, Schwab J M
Department of Microbiology, University of Illinios, Urbana 61801.
J Biol Chem. 1988 Apr 5;263(10):4641-6.
The nucleotide sequence of the fabA gene encoding beta-hydroxydecanoyl thioester dehydrase, a key enzyme of the unsaturated fatty acid synthesis pathway of Escherichia coli, has been determined by the dideoxynucleotide sequencing technique. Most of the sequence was obtained by sequencing intragenic insertions of the transposon, Tn1000, isolated in vivo. A synthetic primer complementary to a portion of the inverted repeat sequences at the ends of the transposon was used to prime DNA synthesis into the flanking fabA sequences. The gene is composed of 516 nucleotides (171 amino acid residues) encoding a protein with a molecular weight of 18,800. Approximately half of the derived amino acid sequence was confirmed by automated Edman sequencing of peptides obtained by cyanogen bromide cleavage. The active site histidine residue (His-70) has been identified by analysis of the peptides labeled by reaction with 14C-labeled 3-decynoyl-N-acetylcysteamine, a specific mechanism-activated inhibitor. A cysteine residue (Cys-69) adjacent to the active site histidine may play the role in catalysis previously assigned to a tyrosine residue. We also report a simplified purification process for the dehydrase beginning with extracts of a brain which greatly overproduces the enzyme.
编码β-羟基癸酰硫酯脱水酶（大肠杆菌不饱和脂肪酸合成途径中的关键酶）的fabA基因的核苷酸序列，已通过双脱氧核苷酸测序技术确定。大部分序列是通过对体内分离的转座子Tn1000的基因内插入片段进行测序获得的。使用与转座子末端一部分反向重复序列互补的合成引物，将DNA合成引导至侧翼的fabA序列中。该基因由516个核苷酸（171个氨基酸残基）组成，编码一种分子量为18,800的蛋白质。通过对溴化氰裂解得到的肽段进行自动埃德曼测序，证实了大约一半的推导氨基酸序列。通过分析与14C标记的3-癸炔酰-N-乙酰半胱胺（一种特异性机制激活抑制剂）反应标记的肽段，鉴定出了活性位点组氨酸残基（His-70）。活性位点组氨酸相邻的半胱氨酸残基（Cys-69）可能在催化作用中发挥先前赋予酪氨酸残基的作用。我们还报告了一种从大脑提取物开始的脱水酶简化纯化方法，大脑提取物能大量过量表达该酶。
