Lessard I A, Domingo G J, Borges A, Perham R N
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, United Kingdom.
Eur J Biochem. 1998 Dec 1;258(2):491-501. doi: 10.1046/j.1432-1327.1998.2580491.x.
Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzyme complex from Bacillus stearothermophilus were overexpressed in Escherichia coli. The E2 component was purified as a large soluble aggregate (molecular mass > 1 x 10(6) Da) with the characteristic 532 symmetry of an icosahedral (60-mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa. The recombinant E2 component in vitro was capable of binding either 60 E3(alpha2) dimers or 60 heterotetramers (alpha2beta2) of the pyruvate decarboxylase (E1) component (also the product of B. stearothermophilus genes overexpressed in E. coli). Assembling the E2 polypeptide chain into the icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit-binding domain in each E2 chain. This has important consequences for the stoichiometry of the assembled complex in vivo. The lipoyl domain of the recombinant E2 protein was found to be unlipoylated, but it could be correctly post-translationally modified in vitro using a recombinant lipoate protein ligase from E. coli. The lipoylated E2 component was able to bind recombinant E1 and E3 components in vitro to generate a PDH complex with a catalytic activity comparable with that of the wild-type enzyme. Reversible unfolding of the recombinant E2 and E3 components in 6 M guanidine hydrochloride was possible in the absence of chaperonins, with recoveries of enzymic activities of 95% and 85%, respectively. However, only 26% of the E1 enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1alpha and E1beta. This represents the first complete post-translational modification and assembly of a fully active PDH complex from recombinant proteins in vitro.
来自嗜热脂肪芽孢杆菌的丙酮酸脱氢酶（PDH）多酶复合体中二氢硫辛酰乙酰转移酶（E2）和二氢硫辛酰脱氢酶（E3）组分的编码基因在大肠杆菌中得到了过表达。E2组分被纯化成为一种大的可溶性聚集体（分子量>1×10⁶ Da），具有二十面体（60聚体）结构的特征性532对称性，而E3则被纯化成为一种分子量为110 kDa的同型二聚体。体外重组E2组分能够结合60个E3（α₂）二聚体或60个丙酮酸脱羧酶（E1）组分的异源四聚体（α₂β₂）（E1也是在大肠杆菌中过表达的嗜热脂肪芽孢杆菌基因的产物）。将E2多肽链组装到二十面体E2核心中，并未对E1或E3与每个E2链中的外周亚基结合结构域的结合造成任何限制。这对于体内组装复合体的化学计量具有重要影响。发现重组E2蛋白的硫辛酰结构域未被硫辛酰化，但使用来自大肠杆菌的重组硫辛酸蛋白连接酶在体外可对其进行正确的翻译后修饰。硫辛酰化的E2组分在体外能够结合重组E1和E3组分，以产生具有与野生型酶相当的催化活性的PDH复合体。在没有伴侣蛋白的情况下，重组E2和E3组分在6 M盐酸胍中能够可逆地展开，酶活性的回收率分别为95%和85%。然而，在相同条件下，由于E1α和E1β的不可逆变性，仅回收了26%的E1酶活性。这代表了首次在体外由重组蛋白完成具有完全活性的PDH复合体的翻译后修饰和组装。
