Naggert J, Narasimhan M L, DeVeaux L, Cho H, Randhawa Z I, Cronan J E, Green B N, Smith S
Children's Hospital Oakland Research Institute, California 94609.
J Biol Chem. 1991 Jun 15;266(17):11044-50.
The gene (tesB) encoding Escherichia coli thioesterase II, a low-abundance enzyme of unknown physiological function which can hydrolyze a broad range of acyl-CoA thioesters, has been localized by transposon mutagenesis, cloned and sequenced. A two-cistron construct containing both the lac and tesB promoters was used successfully to overexpress the 286-residue polypeptide. The recombinant enzyme constituted up to 25% of the soluble proteins of E. coli and was readily purified to homogeneity as a tetramer of approximately 120,000 Da. Amino-terminal sequence analysis and electrospray ionization mass spectrometry confirmed the identity of the thioesterase and revealed that the amino-terminal formyl-methionine had been removed yielding a subunit species of average molecular mass 31,842 Da. The protein does not contain the GXSXG motif found characteristically in animal thioesterases which function as chain-terminating enzymes in fatty acid synthesis and exhibits no sequence similarity with these or any other known proteins. Activity of the recombinant enzyme was inhibited by iodoacetamide and diethylpyrocarbonate. The carboxamidomethylated residue was identified as histidine 58, and a role for this amino acid in catalysis is suggested. E. coli strains having a large deletion within the genomic tesB gene grew normally but retained a low level of thioesterase activity toward decanoyl-CoA. This residual activity indicates the presence of an additional decanoyl-CoA hydrolase in E. coli. Over-expression of the recombinant enzyme, under control of the lac promoter, did not alter the fatty acids synthesized by E. coli at any stage of cell growth and the physiological role of this enzyme remains an enigma.
编码大肠杆菌硫酯酶II的基因（tesB）已通过转座子诱变进行定位、克隆和测序。硫酯酶II是一种低丰度酶，其生理功能未知，但能水解多种酰基辅酶A硫酯。利用含有lac和tesB启动子的双顺反子构建体成功实现了286个氨基酸残基多肽的过表达。重组酶占大肠杆菌可溶性蛋白的比例高达25%，并易于纯化至均一状态，形成约120,000 Da的四聚体。氨基末端序列分析和电喷雾电离质谱法证实了硫酯酶的身份，并表明氨基末端的甲酰甲硫氨酸已被去除，产生平均分子量为31,842 Da的亚基。该蛋白不包含动物硫酯酶中特有的GXSXG基序，动物硫酯酶在脂肪酸合成中起链终止酶的作用，且与这些或任何其他已知蛋白均无序列相似性。重组酶的活性受到碘乙酰胺和焦碳酸二乙酯的抑制。羧甲基化的残基被鉴定为组氨酸58，并推测该氨基酸在催化中起作用。基因组tesB基因内有大片段缺失的大肠杆菌菌株生长正常，但对癸酰辅酶A仍保留低水平的硫酯酶活性。这种残留活性表明大肠杆菌中存在另一种癸酰辅酶A水解酶。在lac启动子的控制下，重组酶的过表达在细胞生长的任何阶段都不会改变大肠杆菌合成的脂肪酸，该酶的生理作用仍然是个谜。
