Stuible H P, Meier S, Schweizer E
Lehrstuhl für Biochemie, Universität Erlangen-Nürnberg, Erlangen, Germany.
Eur J Biochem. 1997 Sep 1;248(2):481-7. doi: 10.1111/j.1432-1033.1997.00481.x.
Upon heterologous expression of the Brevibacterium ammoniagenes type-I fatty acid synthase FAS-A in Escherichia coli, only the pantetheine-free apoenzyme is synthesized. Activation of FAS-A to its holoform was achieved by transformation with a second B. ammoniagenes gene, PPT1, encoding a type-I FAS-specific phosphopantetheine transferase. PPT1 was identified as a coding sequence located immediately downstream of the second FAS gene present on the B. ammoniagenes genome, fasB. Due to this linkage, PPT1 was part of the cloned fasB DNA region and, consequently, FAS-B but not FAS-A was synthesized as holoFAS in E. coli. PPT1 encodes a protein of 153 amino acids and has a calculated molecular mass of 16,884 Da. The PPT1 gene product contains 25% identical and 42% conserved amino acids compared with the type-II acyl-carrier-protein-activating enzyme of E. coli. Although there is essentially no intergenic region between fasB and PPT1, the PPTase gene is autonomously expressed in E. coli if flanked by 200 bp of its endogenous 5' DNA. The structural independence of Ppt1p was confirmed immunologically, as specific antibodies react with the purified PPTase but not with FAS-B. Overexpression and purification of the His-tagged Ppt1p allowed the in vitro activation of apoFAS-A. This holoenzyme synthesis requires, in addition to Ppt1p, CoA and Mg2+ and leads to a specific FAS activity comparable to that of natural B. ammoniagenes FAS-A. The reactivity of the in vitro-activated FAS-A was verified by the optical FAS assay and by analysis of its in vitro products. In agreement with the known overall colinearity of B. ammoniagenes FAS-B and the Saccharomyces cerevisiae FAS1 and FAS2 gene products, a PPT1-like sequence is also observed at the C terminus of FAS2. However, in contrast to B. ammoniagenes PPT1, this sequence is an integral part of the yeast FAS2 gene. Thus, activation of type-I fatty acid synthases may be accomplished by distinct trans-acting PPTase enzymes and by intrinsic cis-acting PPTase domains.
在将产氨短杆菌I型脂肪酸合酶FAS - A在大肠杆菌中进行异源表达时，仅合成了不含泛酰巯基乙胺的脱辅基酶。通过用产氨短杆菌的另一个基因PPT1进行转化，实现了FAS - A向全酶形式的激活，PPT1编码一种I型FAS特异性磷酸泛酰巯基乙胺转移酶。PPT1被鉴定为位于产氨短杆菌基因组上第二个FAS基因fasB下游紧邻的编码序列。由于这种连锁关系，PPT1是克隆的fasB DNA区域的一部分，因此，在大肠杆菌中FAS - B而非FAS - A被合成为全FAS。PPT1编码一个由153个氨基酸组成的蛋白质，计算分子量为16,884 Da。与大肠杆菌的II型酰基载体蛋白激活酶相比，PPT1基因产物含有25%相同和42%保守的氨基酸。尽管fasB和PPT1之间基本上没有基因间区域，但如果PPTase基因两侧有其内源5' DNA的200 bp，它在大肠杆菌中可自主表达。Ppt1p的结构独立性通过免疫学方法得到证实，因为特异性抗体与纯化的PPTase反应，而不与FAS - B反应。His标签的Ppt1p的过表达和纯化使得脱辅基FAS - A能够在体外被激活。这种全酶合成除了需要Ppt1p外，还需要辅酶A和Mg2 +，并产生与天然产氨短杆菌FAS - A相当的特异性FAS活性。通过光学FAS测定法及其体外产物分析验证了体外激活的FAS - A的反应性。与产氨短杆菌FAS - B和酿酒酵母FAS1及FAS2基因产物已知的整体共线性一致，在FAS2的C末端也观察到一个类似PPT1的序列。然而，与产氨短杆菌PPT1不同，该序列是酵母FAS2基因的一个组成部分。因此，I型脂肪酸合酶的激活可能通过不同的反式作用PPTase酶和内在的顺式作用PPTase结构域来完成。
