Machado R S, Clark D P, Guest J R
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
FEMS Microbiol Lett. 1992 Dec 15;100(1-3):243-8. doi: 10.1111/j.1574-6968.1992.tb14047.x.
The lipoate acetyltransferase (E2p) subunits of the pyruvate dehydrogenase (PDH) complex of Escherichia coli have three tandemly repeated lipoyl domains, although net deletions of one or two has no apparent effect on the activity of the purified complexes. Plasmids containing IPTG-inducible aceEF-lpd operons, which encode PDH complexes bearing from one to nine lipoyl domains per E2p chain (24-216 per complex), were constructed. They were all capable of restoring the nutritional lesion of a strain lacking PDH complex and they all expressed active sedimentable multienzyme complexes having a relatively normal range of subunit stoichiometries. The extra domains are presumed to protrude from the E2p core (24-mer) without significantly affecting the assembly of the E1p and E3 subunits on the respective edges and faces of the cubic core. However, the catalytic activities of the overproduced complexes containing four to nine lipoyl domains per E2p chain were lower than those with fewer lipoyl domains. This could be due to under-lipoylation of the domains participating in catalysis and interference from unlipoylated domains.
大肠杆菌丙酮酸脱氢酶（PDH）复合体的硫辛酸乙酰转移酶（E2p）亚基有三个串联重复的硫辛酰结构域，尽管缺失一个或两个结构域对纯化后的复合体活性并无明显影响。构建了含有IPTG诱导型aceEF-lpd操纵子的质粒，该操纵子编码的PDH复合体每个E2p链含有1至9个硫辛酰结构域（每个复合体含24至216个）。它们都能够恢复缺乏PDH复合体的菌株的营养缺陷，并且都表达出具有相对正常亚基化学计量范围的、可沉降的活性多酶复合体。推测额外的结构域从E2p核心（24聚体）突出，而不会显著影响E1p和E3亚基在立方核心各自边缘和表面的组装。然而，每个E2p链含有4至9个硫辛酰结构域的过量表达复合体的催化活性低于含较少硫辛酰结构域的复合体。这可能是由于参与催化的结构域硫辛酰化不足以及未硫辛酰化结构域的干扰所致。
