人源线粒体NAD依赖性异柠檬酸脱氢酶IDH3的结构、功能和调控机制研究

Isocitrate dehydrogenases (IDHs) are a family of enzymes that catalyze the oxidative decarboxylation of isocitrate (ICT) into α-ketoglutarate (α-KG) using NADP or NAD as coenzyme. Mammals including human contain an NADH-IDH (IDH1) in the cytosol and an NADP-IDH (IDH2) in the mitochondrial, and additionally an NAD-IDH (IDH3) in the mitochondrial. IDH3 is composed of three types of subunits in the ratio of 2α:1β:1γ, which are assembled into the αβ and αγ heterodimers which are further assembled into the α2βγ heterotetramer or heterooctamer; and it is an allosteric enzyme whose activity is allosterically regulated positively by CIT and ADP, and negatively by ATP and NADH. IDH3 carries out the catalytic reaction in the tricarboxylic acid (TCA) cycle as the rate-limiting step and thus plays an important role in the metabolism of substance and energy production. However, the structure, catalytic mechanism and regulation mechanism of IDH3 are still largely unknown. In our previous works, we have systematically characterized the enzymatic properties of the αβ and αγ heterodimers and the α2βγ heterotetramer of human IDH3 and performed structural studies of the αγ heterodimer in complexes with the activators CIT and ADP and the inhibitor NADH. The structural and biochemical data together reveal the regulation mechanisms of the αγ heterodimer by CIT/ADP and NADH. In this study, we will carry out further structural and functional studies of the αβ and αγ heterodimers and the α2βγ heterotetramer of human IDH3 using a combination of X-ray crystallography, cryo-electron microscopy, biochemistry and molecular biology, and elucidate the molecular mechanism of the assembly of the αβ and αγ heterodimers into the α2βγ heterotetramer, and the catalytic mechanism, and the allosteric regulation mechanisms of the α2βγ heterotetramer by various activators and inhibitors. The expected results will have important biological significances as well as important biomedical implications.

异柠檬酸脱氢酶（IDH）以NADP或NAD为辅酶、催化异柠檬酸氧化脱羧生成α-酮戊二酸，人含有定位于细胞质的NADP-IDH（IDH1）、以及定位于线粒体的NADP-IDH（IDH2）和NAD-IDH（IDH3），其中IDH3由α2βγ异源四聚体形式存在，其酶活受到ADP、ATP、柠檬酸和NADH等因子的调控。IDH3是参与三羧酸循环的限速酶，在能量产生和合成代谢中发挥十分重要作用，但其结构、催化机理和调控机制还不清楚。我们在前期对IDH3的αβ和αγ二聚体、α2βγ四聚体的生化和酶学性质、以及αγ二聚体的调控机制开展了较为细致的研究，本项目将进一步开展对IDH3的结构和功能研究，通过对不同复合物在不同底物、辅因子和调控因子结合状态下的三维晶体和冷冻电镜结构测定、以及相关复合物的酶学和生化性质研究，系统阐述IDH3中αβ和αγ二聚体的结构和功能、α2βγ四聚体的组装、催化和调节机理。

三羧酸(TCA)循环是有氧生物中一个关键代谢通路，在能量产生和合成代谢中发挥十分重要的功能。人源线粒体定位的NAD-依赖型异柠檬酸脱氢酶(HsIDH3)负责催化异柠檬酸ICT氧化脱羧生成α-KG，这是TCA循环的必要和限速步骤。HsIDH3由α、β、γ三个亚基以2:1:1比例组成，α和β亚基形成αβ异二聚体，α和γ亚基形成αγ异二聚体，αβ和αγ二个异二聚体再组装成α2βγ四聚体，后者再进一步组装成(α2βγ)2八聚体，发挥生物学功能。HsIDH3是一个变构调节酶，其酶活受到众多小分子，包括ADP、ATP、柠檬酸CIT、NADH等的激活和抑制调控。HsIDH3的异常表达或突变与许多疾病有关。.在过去的四年中，我们运用结构生物学、生物化学和分子生物学的方法对HsIDH3的αβ和αγ异二聚体和(α2βγ)2八聚体全酶进行了系统和深入的结构和功能研究。测定了αβ和αγ异二聚体在不结合配体以及结合多种配体的结构; (α2βγ)2全酶在未结合配体状态下的结构；含有α-Q139A突变(αM)、处于组成型激活状态的αMβ和αMγ异二聚体在未结合配体以及结合多种配体的结构，以及(αMβαMγ)2全酶在未结合配体状态下的结构。我们还对活性中心参与辅因子、底物和金属离子结合的关键氨基酸残基，参与组成γ亚基中变构调控位点、β亚基中伪变构调控位点、以及二聚体-二聚体界面的关键氨基酸残基的功能进行了点突变和酶动力学研究。基于结构和功能研究结果，（1）揭示了αβ不受异构激活调控的分子机制、以及受NADH竞争性抑制的分子机制；（2）揭示了ATP对αγ具有激活和抑制双重作用的分子机理；（3）揭示了αβ和αγ组装成α2βγ四聚体、再进一步组装成(α2βγ)2八聚体的分子机制；（4）揭示了别构调控信号在二个异二聚体之间进行传递的分子网络，阐明了(α2βγ)2八聚体的变构激活的分子机制、以及αβ和αγ异二聚体之间发挥协同效应的分子机制；（5）解析了类米氏方程复合物（αQ139AICT+Ca+NADβNAD）的结构，揭示了HsIDH3的催化反应机制。这些研究结果具有重要的生物学意义，同时也为研究高等生物中NAD-IDH酶的结构和功能、催化机制和别构调节机制，以及研究HsIDH3的功能异常与相关疾病发生发展的关系奠定了扎实的分子基础。