Human NAD-dependent isocitrate dehydrogenase or HsIDH3 catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the TCA cycle. HsIDH3 exists and functions as a heterooctamer composed of the αβ and αγ heterodimers, and is regulated allosterically and/or competitively by numerous metabolites including CIT, ADP, ATP, and NADH. In this work, we report the crystal structure of HsIDH3 containing a β mutant in apo form. In the HsIDH3 structure, the αβ and αγ heterodimers form the α₂βγ heterotetramer via their clasp domains, and two α₂βγ heterotetramers form the (α₂βγ)₂ heterooctamer through insertion of the N-terminus of the γ subunit of one heterotetramer into the back cleft of the β subunit of the other heterotetramer. The functional roles of the key residues at the allosteric site, the pseudo allosteric site, the heterodimer and heterodimer–heterodimer interfaces, and the N-terminal of the γ subunit are validated by mutagenesis and kinetic studies. Our structural and biochemical data together demonstrate that the allosteric site plays an important role but the pseudo allosteric site plays no role in the allosteric activation of the enzyme; the activation signal from the allosteric site is transmitted to the active sites of both αβ and αγ heterodimers via the clasp domains; and the N-terminal of the γ subunit plays a critical role in the formation of the heterooctamer to ensure the optimal activity of the enzyme. These findings reveal the molecular mechanism of the assembly and allosteric regulation of HsIDH3.

人NAD依赖性异柠檬酸脱氢酶或HsIDH3在TCA循环中催化异柠檬酸脱羧为α-酮戊二酸。HsIDH3存在并充当由αβ和αγ异二聚体组成的杂八聚体，并受多种代谢物（包括CIT，ADP，ATP和NADH）的变构和/或竞争性调节。在这项工作中，我们报告了含有apo形式的β突变体的HsIDH3的晶体结构。在HsIDH3结构中，αβ和αγ异二聚体形成α ₂经由其扣域βγ异四聚体，和两个α ₂ βγ异源四聚形成（α ₂ βγ）₂通过将一个异四聚体的γ亚基的N端插入另一个异四聚体的β亚基的后半裂中来形成异八聚体。通过诱变和动力学研究验证了变构位点，假变构位点，异二聚体和异二聚体-异二聚体界面以及γ亚基的N端关键残基的功能作用。我们的结构和生化数据一起证明，变构位点起着重要的作用，但假变构位点在酶的变构活化中不起作用。来自变构位点的激活信号通过扣结构域传递至αβ和αγ异二聚体的活性位点；γ亚基的N端在杂八聚体的形成中起关键作用，以确保酶的最佳活性。这些发现揭示了HsIDH3的组装和变构调节的分子机制。