Tsay J T, Oh W, Larson T J, Jackowski S, Rock C O
Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
J Biol Chem. 1992 Apr 5;267(10):6807-14.
beta-Ketoacyl-acyl carrier protein (ACP) synthase III catalyzes the condensation of acetyl-CoA with malonyl-ACP in dissociated (Type II) fatty acid synthase systems. A synthase III mutant was used to localize the structural gene to the 24.5-min region of the Escherichia coli chromosome, and the defective synthase III allele was designated fabH1. The fabH gene was identified on a 1.3-kilobase NruI-HindIII chromosomal DNA fragment (plasmid pWO114) that complemented the enzymatic defect in fabH1 strains. The NruI-HindIII fragment was sequenced and contained a single open reading frame predicted to encode a 33,517-dalton protein with an isoelectric point of 4.85. The fabH sequence contained an Ala-Cys-Ala tripeptide characteristic of condensing enzyme active sites. A T7 expression system showed that the NruI-HindIII fragment directed the synthesis of a single 34,800-dalton protein. This protein was purified and the order of the amino-terminal 30 residues of the protein corresponded exactly to the amino acid structure predicted from the DNA sequence. The purified protein possessed both acetoacetyl-ACP synthase and acetyl-CoA:ACP transacylase activities, and cells harboring plasmid pWO114 overproduced the two activities, supporting the conclusion that a single protein carries out both reactions. Overproduction of synthase III resulted in a significant increase in shorter-chain fatty acids in the membrane phospholipids. These catalytic properties are consistent with the proposed role of synthase III in the initiation of fatty acid synthesis.
β-酮脂酰-酰基载体蛋白（ACP）合酶III在解离型（II型）脂肪酸合酶系统中催化乙酰辅酶A与丙二酸单酰-ACP的缩合反应。利用一个合酶III突变体将结构基因定位到大肠杆菌染色体的24.5分钟区域，有缺陷的合酶III等位基因被命名为fabH1。fabH基因在一个1.3千碱基的NruI-HindIII染色体DNA片段（质粒pWO114）上被鉴定出来，该片段能互补fabH1菌株中的酶缺陷。对NruI-HindIII片段进行了测序，其包含一个单一的开放阅读框，预计编码一个33517道尔顿的蛋白质，等电点为4.85。fabH序列包含一个缩合酶活性位点特有的丙氨酸-半胱氨酸-丙氨酸三肽。一个T7表达系统表明，NruI-HindIII片段指导合成一种单一的34800道尔顿的蛋白质。该蛋白质被纯化，其氨基末端30个残基的顺序与从DNA序列预测的氨基酸结构完全一致。纯化后的蛋白质同时具有乙酰乙酰-ACP合酶和乙酰辅酶A：ACP转酰基酶活性，携带质粒pWO114的细胞过量产生这两种活性，支持了单一蛋白质执行这两种反应的结论。合酶III的过量表达导致膜磷脂中短链脂肪酸显著增加。这些催化特性与合酶III在脂肪酸合成起始中所提出的作用一致。
