Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase.
Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase.
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DOI：
10.1016/s0021-9258(19)74070-8
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发表时间：
1992-12
期刊：
影响因子：
--
通讯作者：
P. Black;C. DiRusso;A. Metzger;Tamra L. Heimert
中科院分区：
文献类型：
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作者：
P. Black;C. DiRusso;A. Metzger;Tamra L. Heimert
在肠道细菌大肠杆菌中，酰基辅酶A合成酶（脂肪酸：CoA连接酶（AMP形成）EC 6.2.1.3）激活外源性长链脂肪酸，伴随着它们穿过内膜转运成代谢活性的CoA硫酯。这些化合物在β-氧化过程的第一步中作为酰基辅酶A脱氢酶的底物。已在Kohara E. coli基因文库中，并通过亚克隆和互补分析的方法，显示包含在基因组DNA的2.2-脱氢酶NcoI-ClaI片段上。在T7 RNA聚合酶依赖性诱导后鉴定了该DNA片段内编码的多肽，并使用SDS-聚丙烯酰胺凝胶电泳估计其M（r）= 62，000。通过自动测序确定酰基辅酶A合成酶的N-末端氨基酸序列为Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro。2.2-丝氨酸蛋白酶NcoI-ClaI片段的序列分析揭示了编码这些氨基酸的单个开放阅读框作为分子量为62，028的蛋白质的前10个残基。甲硫氨酸的起始密码子是TTG。从两个fadD特异性寡核苷酸的体内总mRNA的引物延伸定义了在预测的翻译起始位点上游60个碱基对的腺嘌呤残基处的转录起始。通过DNA酶I足迹法在位置-13至-29（OD 1）和位置-99至-115（OD 2）鉴定了fadD基因的两个FadR操纵位点。将E.大肠杆菌酰基辅酶A合成酶与大鼠和酵母酰基辅酶A合成酶以及萤火虫荧光素酶的推导氨基酸序列的比较表明，这些酶具有很大程度的相似性。基于这四种酶相似的反应机制，这种相似性可以定义相同功能所需的区域。
In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of beta-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2-kilobase NcoI-ClaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be M(r) = 62,000 using SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of acyl-CoA synthetase was determined by automated sequencing to be Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro. Sequence analysis of the 2.2-kilobase NcoI-ClaI fragment revealed a single open reading frame encoding these amino acids as the first 10 residues of a protein with a molecular weight of 62,028. The initiation codon for methionine was TTG. Primer extension of total in vivo mRNA from two fadD-specific oligonucleotides defined the transcriptional start at an adenine residue 60 base pairs upstream from the predicted translational start site. Two FadR operator sites of the fadD gene were identified at positions -13 to -29 (OD1) and positions -99 to -115 (OD2) by DNase I footprinting. Comparisons of the predicted amino acid sequence of the E. coli acyl-CoA synthetase to the deduced amino acid sequences of the rat and yeast acyl-CoA synthetases and the firefly luciferase demonstrated that these enzymes shared a significant degree of similarity. Based on the similar reaction mechanisms of these four enzymes, this similarity may define a region required for the same function.