Abstract

Human NAD-dependent isocitrate dehydrogenase or IDH3 catalyzes the decarboxylation of isocitrate into -ketoglutarate in the TCA cycle. We here report the structure of the IDH3 holoenzyme, in which the {beta} and {gamma} heterodimers assemble the 2{beta}{gamma} heterotetramer via their clasp domains, and two 2{beta}{gamma} heterotetramers assemble the (2{beta}{gamma})2 heterooctamer via the {beta} and {gamma} subunits. The functional roles of the key residues involved in the assembly and allosteric regulation are validated by mutagenesis and kinetic studies. The allosteric site plays an important role but the pseudo allosteric site plays no role in the allosteric activation; the activation signal from the allosteric site is transmitted to the active sites of both heterodimers via the clasp domains; and the N-terminus of the {gamma} subunit plays a critical role in the formation and function of the holoenzyme. These findings reveal the molecular mechanism of the assembly and allosteric regulation of human IDH3 holoenzyme.