Ali S T, Guest J R
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, U.K.
Biochem J. 1990 Oct 1;271(1):139-45. doi: 10.1042/bj2710139.
The dihydrolipoamide acetyltransferase subunit (E2p) of the pyruvate dehydrogenase complex of Escherichia coli has three highly conserved and tandemly repeated lipoyl domains, each containing approx. 80 amino acid residues. These domains are covalently modified with lipoyl groups bound in amide linkage to the N6-amino groups of specific lysine residues, and the cofactors perform essential roles in the formation and transfer of acetyl groups by the dehydrogenase (E1p) and acetyltransferase (E2p) subunits. A subgene encoding a hybrid lipoyl domain was previously shown to generate two products when overexpressed, whereas a mutant subgene, in which the lipoyl-lysine codon is replaced by a glutamine codon, expresses only one product. A method has been devised for purifying the three types of independently folded domain from crude extracts of E. coli, based on their pH-(and heat-)stabilities. The domains were characterized by: amino acid and N-terminal sequence analysis, lipoic acid content, acetylation by E1p, tryptic peptide analysis and immunochemical activity. This has shown that the two forms of domain expressed from the parental subgene are lipoylated (L203) and unlipoylated (U203) derivatives of the hybrid lipoyl domain, whereas the mutant subgene produces a single unlipoylatable domain (204) containing the Lys-244----Gln substitution.
大肠杆菌丙酮酸脱氢酶复合体的二氢硫辛酰胺乙酰转移酶亚基（E2p）有三个高度保守且串联重复的硫辛酰结构域，每个结构域约含80个氨基酸残基。这些结构域通过与特定赖氨酸残基的N6-氨基以酰胺键结合的硫辛酰基团进行共价修饰，并且这些辅因子在脱氢酶（E1p）和乙酰转移酶（E2p）亚基形成和转移乙酰基的过程中发挥着重要作用。先前研究表明，编码杂合硫辛酰结构域的亚基因在过表达时会产生两种产物，而一个硫辛酰赖氨酸密码子被谷氨酰胺密码子取代的突变亚基因仅表达一种产物。基于三种独立折叠结构域的pH稳定性（和热稳定性），已设计出一种从大肠杆菌粗提物中纯化它们的方法。这些结构域通过以下方法进行表征：氨基酸和N端序列分析、硫辛酸含量、E1p的乙酰化、胰蛋白酶肽段分析和免疫化学活性。结果表明，从亲本亚基因表达的两种形式的结构域是杂合硫辛酰结构域的硫辛酰化（L203）和未硫辛酰化（U203）衍生物，而突变亚基因产生一个单一的不可硫辛酰化结构域（204），其中含有Lys-244→Gln替换。
