Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Japan.
Int J Biol Sci. 2010 Dec 30;7(1):9-17. doi: 10.7150/ijbs.7.9.
Acetoacetyl-CoA thiolase (AT) is an enzyme that catalyses the CoA-dependent thiolytic cleavage of acetoacetyl-CoA to yield 2 molecules of acetyl-CoA, or the reverse condensation reaction. A full-length cDNA clone pBSGT-3, which has homology to known thiolases, was isolated from Dictyostelium cDNA library. Expression of the protein encoded in pBSGT-3 in Escherichia coli, its thiolase enzyme activity, and the amino acid sequence homology search revealed that pBSGT-3 encodes an AT. The recombinant AT (r-thiolase) was expressed in an active form in an E. coli expression system, and purified to homogeneity by selective ammonium sulfate fractionation and two steps of column chromatography. The purified enzyme exhibited a specific activity of 4.70 mU/mg protein. Its N-terminal sequence was (NH₂)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn-Leu-Glu-, which corresponds to the sequence from positions 15 to 24 of the amino acid sequence deduced from pBSGT-3 clone. The r-thiolase in the inclusion body expressed highly in E. coli was the precursor form, which is slightly larger than the purified r-thiolase. When incubated with the cell-free extract of Dictyostelium cells, the precursor was converted to the same size to the purified r-thiolase, suggesting that the presequence at the N-terminus is removed by a Dictyostelium processing peptidase.
乙酰乙酰辅酶 A 硫解酶 (AT) 是一种酶，能够催化辅酶 A 依赖的硫解裂解乙酰乙酰辅酶 A 生成 2 个乙酰辅酶 A 分子，或者进行相反的缩合反应。从粘菌 cDNA 文库中分离出全长 cDNA 克隆 pBSGT-3，该克隆与已知的硫解酶具有同源性。pBSGT-3 编码的蛋白在大肠杆菌中的表达、其硫解酶活性以及氨基酸序列同源性搜索表明，pBSGT-3 编码 AT。重组 AT（r-硫解酶）以活性形式在大肠杆菌表达系统中表达，并通过选择性硫酸铵分级沉淀和两步柱层析纯化至均一性。纯化的酶表现出 4.70 mU/mg 蛋白的比活性。其 N 端序列为 (NH₂)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn-Leu-Glu-，与从 pBSGT-3 克隆推导的氨基酸序列第 15 位到第 24 位的序列相对应。大肠杆菌中高表达的包涵体中的 r-硫解酶是前体形式，比纯化的 r-硫解酶略大。当与粘菌细胞的无细胞提取物孵育时，前体转化为与纯化的 r-硫解酶相同大小的形式，表明 N 端的前导序列被粘菌加工肽酶切除。
