Raasch Katharina, Bocola Marco, Labahn Jörg, Leitner Alexander, Eggeling Lothar, Bott Michael
Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, Jülich, Germany.
Institute of Biotechnology, RWTH Aachen University, Aachen, Germany.
J Biotechnol. 2014 Dec 10;191:99-105. doi: 10.1016/j.jbiotec.2014.05.023. Epub 2014 Jun 4.
Pyruvate dehydrogenase and oxoglutarate dehydrogenase catalyze key reactions in central metabolism. In Corynebacterium glutamicum and related bacteria like Mycobacterium tuberculosis both activities reside in a novel protein supercomplex with the fusion protein OdhA catalyzing the conversion of oxoglutarate to succinyl-coenzyme A. This activity is inhibited by the forkhead-associated (FHA) domain of the small autoinhibitory protein OdhI. Here we used a biological screen which enabled us to isolate suppressor mutants that are influenced in OdhA-OdhI interaction. Five mutants carrying an OdhI mutation were isolated and one with an OdhA mutation. The OdhA mutein OdhA-C704E and three additional C704 variants were constructed. They exhibited unaltered or even slightly enhanced OdhA activity but showed reduced inhibition and interaction with OdhI. The FHA domain of OdhI was crystallized and its structure found in full agreement with previously determined NMR structures. Based on further structural studies, OdhA-OdhI crosslinking experiments, and modeling we discuss the experimental data generated on OdhA-OdhI interaction, with the latter protein representing a rare example of an FHA domain also recognizing a non-phosphorylated interaction partner.
丙酮酸脱氢酶和氧代戊二酸脱氢酶催化中心代谢中的关键反应。在谷氨酸棒杆菌及相关细菌如结核分枝杆菌中，这两种活性存在于一种新型蛋白质超复合物中，其中融合蛋白OdhA催化氧代戊二酸转化为琥珀酰辅酶A。这种活性受到小的自抑制蛋白OdhI的叉头相关（FHA）结构域的抑制。在此，我们采用了一种生物学筛选方法，使我们能够分离出在OdhA - OdhI相互作用中受到影响的抑制突变体。分离出了五个携带OdhI突变的突变体和一个携带OdhA突变的突变体。构建了OdhA突变蛋白OdhA - C704E以及另外三个C704变体。它们表现出未改变甚至略有增强的OdhA活性，但与OdhI的抑制作用和相互作用有所降低。OdhI的FHA结构域被结晶，其结构与先前确定的核磁共振结构完全一致。基于进一步的结构研究、OdhA - OdhI交联实验和建模，我们讨论了关于OdhA - OdhI相互作用产生的实验数据，后者蛋白代表了FHA结构域识别非磷酸化相互作用伙伴的罕见例子。
