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Abstract
Mammalian mitochondrial NAD-dependent isocitrate dehydrogenase (NAD-IDH) catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the tricarboxylic acid cycle. It exists as the α2βγ heterotetramer composed of the αβ and αγ heterodimers. Different from the αγ heterodimer that can be allosterically activated by CIT and ADP, the αβ heterodimer cannot be allosterically regulated by the activators; however, the molecular mechanism is unclear. We report here the crystal structures of the αβ heterodimer of human NAD-IDH with the α subunit in apo form and in Ca2+-bound, NAD-bound, and NADH-bound forms. Structural analyses and comparisons reveal that the αβ heterodimer has a similar yet more compact overall structure compared with the αγ heterodimer and contains a pseudo-allosteric site that is structurally different from the allosteric site. In particular, the β3-α3 and β12-α8 loops of the β subunit at the pseudo-allosteric site adopt significantly different conformations from those of the γ subunit at the allosteric site and hence impede the binding of the activators, explaining why the αβ heterodimer cannot be allosterically regulated by the activators. The structural data also show that NADH can compete with NAD to bind to the active site and inhibits the activity of the αβ heterodimer. These findings together with the biochemical data reveal the molecular basis for the function of the αβ heterodimer of human NAD-IDH.
Highlights
Mammalian mitochondrial NAD-dependent isocitrate dehydrogenase (NAD-isocitrate dehydrogenases (IDHs)) catalyzes the decarboxylation of isocitrate into ␣-ketoglutarate in the tricarboxylic acid cycle
Eukaryotic cells contain both NADP-IDHs and NAD-dependent IDHs (NAD-IDHs); the NAD-dependent isocitrate dehydrogenase (NAD-IDH) localized in the mitochondria catalyze the reaction in the tricarboxylic acid (TCA) cycle, and the NADP-IDHs localized in the cytosol and mitochondria play important roles in cellular defense against oxidative damage, detoxification of reactive oxygen species, and synthesis of fat and cholesterol [1,2,3,4]
Sequence analysis shows that the C-terminal regions of the ␣, , and ␥ subunits of human NAD-IDH are substantially different from each other and varied among different species (Fig. S1)
Summary
Mammalian mitochondrial NAD-dependent isocitrate dehydrogenase (NAD-IDH) catalyzes the decarboxylation of isocitrate into ␣-ketoglutarate in the tricarboxylic acid cycle. The structural data show that NADH can compete with NAD to bind to the active site and inhibits the activity of the ␣ heterodimer These findings together with the biochemical data reveal the molecular basis for the function of the ␣ heterodimer of human NAD-IDH. Eukaryotic cells contain both NADP-IDHs and NAD-dependent IDHs (NAD-IDHs); the NAD-IDHs localized in the mitochondria catalyze the reaction in the TCA cycle, and the NADP-IDHs localized in the cytosol and mitochondria play important roles in cellular defense against oxidative damage, detoxification of reactive oxygen species, and synthesis of fat and cholesterol [1,2,3,4]. The functional, structural, and mechanistic studies of both types of IDHs have important biological and biomedical significance
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