Isolation and characterization of the beta-ketoacyl-acyl carrier protein synthase III gene (fabH) from Escherichia coli K-12.
Isolation and characterization of the beta-ketoacyl-acyl carrier protein synthase III gene (fabH) from Escherichia coli K-12.
复制标题
DOI：
10.1016/s0021-9258(19)50498-7
复制
发表时间：
1992-04
期刊：
影响因子：
--
通讯作者：
J. Tsay;W. Oh;T. Larson;S. Jackowski;C. Rock
中科院分区：
文献类型：
--
作者：
J. Tsay;W. Oh;T. Larson;S. Jackowski;C. Rock
β-酮酰基-酰基载体蛋白（ACP）合酶III催化乙酰辅酶A与丙二酰-ACP在解离的（II型）脂肪酸合酶系统中的缩合。一个合成酶III突变体被用来本地化的结构基因的大肠杆菌染色体的24.5分钟的区域，和有缺陷的合成酶III等位基因被指定为fabH 1。在1.3-脱氢酶NruI-HindIII染色体DNA片段（质粒pWO 114）上鉴定了fabH基因，该片段补充了fabH 1菌株中的酶缺陷。对NruI-HindIII片段进行测序，该片段含有一个预测编码33，517-道尔顿蛋白质的单一开放阅读框，等电点为4.85。fabH序列含有一个Ala-Cys-Ala三肽，具有缩合酶活性位点的特征。T7表达系统显示NruI-HindIII片段指导单个34，800-道尔顿蛋白的合成。该蛋白质被纯化，并且该蛋白质的氨基末端30个残基的顺序完全对应于从DNA序列预测的氨基酸结构。纯化的蛋白质具有乙酰乙酰-ACP合酶和乙酰-CoA：ACP转酰酶活性，并且携带质粒pWO 114的细胞过量产生这两种活性，支持单一蛋白质进行两种反应的结论。合成酶III的过度生产导致膜磷脂中短链脂肪酸的显着增加。这些催化特性与合成酶III在脂肪酸合成起始中的拟议作用一致。
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.