Formation of reactive oxygen species by human and bacterial pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes reconstituted from recombinant components.
Formation of reactive oxygen species by human and bacterial pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes reconstituted from recombinant components.
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DOI：
10.1016/j.freeradbiomed.2015.10.001
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发表时间：
2015-12
影响因子：
7.4
通讯作者：
Adam-Vizi V
中科院分区：
文献类型：
--
作者：
Ambrus A;Nemeria NS;Torocsik B;Tretter L;Nilsson M;Jordan F;Adam-Vizi V
人和大肠杆菌（Escherichia coli）的丙酮酸和2-酮戊二酸脱氢酶多酶复合物（PDHc，OGDHc）的单个重组组分。coli）来源的大肠杆菌中表达并纯化。coli中进行了优化。四种多酶复合物各自在最佳条件下以不同的化学计量比重构。结合化学计量的最高催化效率，确定从NADH生成的络合物在生理pH值的速率。由于这些络合物中的一些被证明具有“兼职”活动的病理条件下，往往伴随着酸中毒，活动也被确定在pH值为6.3。由于hOGDHc的E3组分产生活性氧（ROS）是一种病理相关特征，因此通过乙酰化细胞色素c还原法在正向和反向催化方向上测量具有最佳化学计量的复合物产生的超氧化物。研究了各种已知的生理活性和ROS产生的影响因素，包括Ca ~（2+）、ADP、脂酰化状态或pH。人复合物也与E3组分的最普遍的人病理突变体G194 C重构并表征;先前报道具有G194 C取代的分离的人E3具有增强的ROS产生能力。结果表明：i. PDHc与OGDHc一样，也能产生活性氧，这一特性在大肠杆菌和大肠杆菌中都表现出来。大肠杆菌和人复合物，ii.当ROS生成是针对其共同E3组分的单位质量计算时，重构的hPDHc在正向和反向反应中都以与hOGDHc相比显著更高的速率生成ROS，iii. E1组分或E1-E2亚复合物仅在hOGDHc中产生显著量的ROS; iv.将hE 3的G194 C变体（致病突变的结果）掺入重建的hOGDHc和hPDHc中确实导致两种复合物的活性降低，并且仅通过hOGDHc和仅在其逆反应中产生更高的ROS。
Individual recombinant components of pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes (PDHc, OGDHc) of human and Escherichia coli (E. coli) origin were expressed and purified from E. coli with optimized protocols. The four multienzyme complexes were each reconstituted under optimal conditions at different stoichiometric ratios. Binding stoichiometries for the highest catalytic efficiency were determined from the rate of NADH generation by the complexes at physiological pH. Since some of these complexes were shown to possess ‘moonlighting’ activities under pathological conditions often accompanied by acidosis, activities were also determined at pH 6.3. As reactive oxygen species (ROS) generation by the E3 component of hOGDHc is a pathologically relevant feature, superoxide generation by the complexes with optimal stoichiometry was measured by the acetylated cytochrome c reduction method in both the forward and the reverse catalytic directions. Various known affectors of physiological activity and ROS production, including Ca2+, ADP, lipoylation status or pH, were investigated. The human complexes were also reconstituted with the most prevalent human pathological mutant of the E3 component, G194C and characterized; isolated human E3 with the G194C substitution was previously reported to have an enhanced ROS generating capacity. It is demonstrated that: i. PDHc, similarly to OGDHc, is able to generate ROS and this feature is displayed by both the E. coli and human complexes, ii. Reconstituted hPDHc generates ROS at a significantly higher rate as compared to hOGDHc in both the forward and the reverse reactions when ROS generation is calculated for unit mass of their common E3 component, iii. The E1 component or E1-E2 subcomplex generates significant amount of ROS only in hOGDHc; iv. Incorporation of the G194C variant of hE3, the result of a disease-causing mutation, into reconstituted hOGDHc and hPDHc indeed leads to a decreased activity of both complexes and higher ROS generation by only hOGDHc and only in its reverse reaction.