Rat long chain acyl-CoA synthetase 5, but not 1, 2, 3, or 4, complements Escherichia coli fadD
Rat long chain acyl-CoA synthetase 5, but not 1, 2, 3, or 4, complements Escherichia coli fadD
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DOI：
10.1074/jbc.m311392200
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发表时间：
2004-03-19
影响因子：
4.8
通讯作者：
Lewin, TM
中科院分区：
文献类型：
--
作者：
Caviglia, JM;Li, LO;Lewin, TM
长链脂肪酸通过酰基辅酶A合成酶（脂肪酸辅酶A连接酶：AMP形成，E.C.6.2.1.3；ACS）转化为酰基辅酶A。大肠杆菌有一个 ACS，即 FadD，当脂肪酸是唯一的碳和能量来源时，它对于生长至关重要。啮齿动物有五种 ACS 亚型，它们在底物特异性、组织表达和亚细胞定位方面有所不同，并且被认为将脂肪酸引导至不同的代谢途径。我们在缺乏 FadD 的大肠杆菌菌株中表达了大鼠 ACS 亚型 1 - 5。所有大鼠 ACS 亚型均在大肠杆菌 fadD 或 fadDfadR 中表达，并且具有比表达 FadD 的野生型对照菌株高 1.6-20 倍的 ACS 比活性。在fadD背景中，大鼠ACS亚型1、2、3、4和5以野生型水平的5%至25%氧化[C-14]油酸，但只有ACS5以油酸作为唯一碳源恢复生长。为了确保β-氧化酶不受限制，还在fadD fadR菌株中检查了ACS活性、β-氧化、脂肪酸转运和磷脂合成的测定，从而消除了FadR对转运蛋白FadL和β-氧化酶的抑制。在该菌株中，ACS1、2、3 和 4 的脂肪酸转运水平较低，但可检测到，并且 ACS5 的脂肪酸转运水平接近野生型水平的 50%。尽管β-氧化增加，但只有ACS5转化体能够在油酸上生长。这些研究表明，尽管 ACS 亚型 1 - 4 不同程度地支持中等转运活性、β-氧化和磷脂合成，并且尽管它们的体外比活性大于染色体编码的 FadD，但它们在生长方面无法在功能上替代 FadD。因此，膜组成和蛋白质-蛋白质相互作用对于重建细菌 ACS 功能可能至关重要。
Long chain fatty acids are converted to acyl-CoAs by acyl-CoA synthetase ( fatty acid CoA ligase: AMP forming, E. C. 6.2.1.3; ACS). Escherichia coli has a single ACS, FadD, that is essential for growth when fatty acids are the sole carbon and energy source. Rodents have five ACS isoforms that differ in substrate specificity, tissue expression, and subcellular localization and are believed to channel fatty acids toward distinct metabolic pathways. We expressed rat ACS isoforms 1 - 5 in an E. coli strain that lacked FadD. All rat ACS isoforms were expressed in E. coli fadD or fadDfadR and had ACS specific activities that were 1.6-20-fold higher than the wild type control strain expressing FadD. In the fadD background, the rat ACS isoforms 1, 2, 3, 4 and 5 oxidized [C-14] oleate at 5 to 25% of the wild type levels, but only ACS5 restored growth on oleate as the sole carbon source. To ensure that enzymes of beta-oxidation were not limiting, assays of ACS activity, beta-oxidation, fatty acid transport, and phospholipid synthesis were also examined in a fadD fadR strain, thereby eliminating FadR repression of the transporter FadL and the enzymes of beta-oxidation. In this strain, fatty acid transport levels were low but detectable for ACS1, 2, 3, and 4 and were nearly 50% of wild type levels for ACS5. Despite increases in beta-oxidation, only ACS5 transformants were able to grow on oleate. These studies show that although ACS isoforms 1 - 4 variably supported moderate transport activity, beta-oxidation, and phospholipid synthesis and although their in vitro specific activities were greater than that of chromosomally encoded FadD, they were unable to substitute functionally for FadD regarding growth. Thus, membrane composition and protein-protein interactions may be critical in reconstituting bacterial ACS function.