Pazirandeh M, Chirala S S, Huang W Y, Wakil S J
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1989 Oct 25;264(30):18195-201.
Fatty acid synthase of animal tissue is a multifunctional enzyme comprised of two identical subunits, each containing seven partial activities and a site for the prosthetic group, 4'-phosphopantetheine (acyl carrier protein). We have recently isolated cDNA clones of chicken fatty acid synthase coding for the dehydratase, enoyl reductase, beta-ketoacyl reductase, acyl carrier protein, and thioesterase domains (Chirala, S.S., Kasturi, R., Pazirandeh, M., Stolow, D.T., Huang, W.Y., and Wakil, S.J. (1989) J. Biol. Chem. 264, 3750-3757). To gain insight into the structure and function of the various domains, the portion of the cDNA coding for the acyl carrier protein and thioesterase domains was expressed in Escherichia coli by using an expression vector that utilizes the phage lambda PL promoter. The recombinant protein was efficiently expressed and purified to near homogeneity using anion-exchange and hydroxyapatite chromatography. As expected from the coding capacity of the cDNA expressed, the protein has a molecular weight of 43,000 and reacts with antithioesterase antibodies. The recombinant thioesterase was found to be enzymatically active and has the same substrate specificity and kinetic properties as the native enzyme of the multifunctional synthase. Treatment of the recombinant protein with alpha-chymotrypsin results in the cleavage of the acyl carrier protein and thioesterase domain junction sequence at exactly the same site as with native fatty acid synthase. The amino acid composition of the purified recombinant protein revealed the presence of 0.6 mol of beta-alanine/mol of protein, indicating partial pantothenylation of the recombinant acyl carrier protein domain. These results indicate that the expressed protein has a conformation similar to the native enzyme and that its folding into functionally active domains is independent of the remaining domains of the multifunctional synthase subunit. These conclusions are consistent with the proposal that the multifunctional synthase gene has evolved from fusion of component genes.
动物组织中的脂肪酸合酶是一种多功能酶，由两个相同的亚基组成，每个亚基包含七种部分活性以及一个辅基4'-磷酸泛酰巯基乙胺（酰基载体蛋白）的位点。我们最近分离出了鸡脂肪酸合酶的cDNA克隆，其编码脱水酶、烯酰还原酶、β-酮酰还原酶、酰基载体蛋白和硫酯酶结构域（奇拉拉，S.S.，卡斯图里，R.，帕齐兰德，M.，斯托洛，D.T.，黄，W.Y.，和瓦基尔，S.J.（1989年）《生物化学杂志》264，3750 - 3757）。为深入了解各个结构域的结构和功能，利用一个使用噬菌体λ PL启动子的表达载体，将编码酰基载体蛋白和硫酯酶结构域的cDNA部分在大肠杆菌中进行表达。重组蛋白得到高效表达，并通过阴离子交换和羟基磷灰石色谱法纯化至近乎均一。正如所表达的cDNA的编码能力所预期的那样，该蛋白分子量为43,000，并且能与抗硫酯酶抗体发生反应。发现重组硫酯酶具有酶活性，并且具有与多功能合酶的天然酶相同的底物特异性和动力学性质。用α-胰凝乳蛋白酶处理重组蛋白会导致酰基载体蛋白和硫酯酶结构域连接序列在与天然脂肪酸合酶完全相同的位点处断裂。纯化的重组蛋白的氨基酸组成显示每摩尔蛋白含有0.6摩尔的β-丙氨酸，这表明重组酰基载体蛋白结构域发生了部分泛酰化。这些结果表明所表达的蛋白具有与天然酶相似的构象，并且其折叠成功能活性结构域与多功能合酶亚基的其余结构域无关。这些结论与多功能合酶基因是由组成基因融合进化而来的提议一致。
