Stuible H P, Meier S, Wagner C, Hannappel E, Schweizer E
Institut für Mikrobiologie, Biochemie und Genetik, Universität Erlangen-Nürnberg, D-91058 Erlangen, Germany.
J Biol Chem. 1998 Aug 28;273(35):22334-9. doi: 10.1074/jbc.273.35.22334.
In Saccharomyces cerevisiae, the low molecular weight acyl carrier protein (ACP) of mitochondrial type II fatty acid synthase (FAS) and the cytoplasmic type I FAS multienzyme contain 4'-phosphopantetheine as a prosthetic group. Sequence alignment studies with the recently isolated phosphopantetheine:protein transferase (PPTase), Ppt1p, from Brevibacterium ammoniagenes revealed the yeast open reading frame, YPL148C, as a potential PPTase gene (25% identical and 43% conserved amino acids). In accordance with this similarity, pantetheinylation of mitochondrial ACP was lost upon disruption of YPL148C. In contrast, biosynthesis of cytoplasmic holo-FAS remained unaffected by this mutation. According to these characteristics, the newly identified gene was designated as PPT2. Similar to ACP null mutants, cellular lipoic acid synthesis and, hence, respiration were abolished in PPT2 deletants. ACP pantetheinylation, lipoic acid synthesis, and respiratory competence were restored upon transformation of PPT2 mutants with cloned PPT2 DNA. In vitro, holo-ACP synthesis was achieved by incubating apo-ACP with coenzyme A in the presence of purified Ppt2p. The homologous yeast enzyme could be replaced, in this assay, by the ACP synthase (EC 2.7.8.7) of Escherichia coli but not by the type I FAS-specific PPTase of B. ammoniagenes, Ppt1p. These results conform with the inability of Ppt2p to activate the cytoplasmic type I FAS complex of yeast.
在酿酒酵母中，线粒体II型脂肪酸合酶（FAS）的低分子量酰基载体蛋白（ACP）和细胞质I型FAS多酶含有4'-磷酸泛酰巯基乙胺作为辅基。对最近从产氨短杆菌中分离出的磷酸泛酰巯基乙胺：蛋白质转移酶（PPTase）Ppt1p进行的序列比对研究表明，酵母开放阅读框YPL148C是一个潜在的PPTase基因（氨基酸序列一致性为25%，保守性为43%）。根据这种相似性，YPL148C被破坏后，线粒体ACP的泛酰化作用丧失。相比之下，细胞质全酶FAS的生物合成不受该突变的影响。根据这些特征，新鉴定出的基因被命名为PPT2。与ACP缺失突变体相似，PPT2缺失突变体中细胞硫辛酸合成以及呼吸作用被消除。用克隆的PPT2 DNA转化PPT2突变体后，ACP泛酰化、硫辛酸合成和呼吸能力得以恢复。在体外，通过在纯化的Ppt2p存在下将脱辅基ACP与辅酶A一起孵育可实现全酶ACP的合成。在此测定中，该酵母同源酶可被大肠杆菌的ACP合酶（EC 2.7.8.7）替代，但不能被产氨短杆菌的I型FAS特异性PPTase Ppt1p替代。这些结果与Ppt2p无法激活酵母细胞质I型FAS复合物的情况相符。
