Allosteric Motions in Structures of Yeast NAD+-specific Isocitrate Dehydrogenase

Alexander B Taylor,Gang Hu,P John Hart,Lee Mcalister-Henn

doi:10.1074/jbc.m708719200

Abstract

Mitochondrial NAD(+)-specific isocitrate dehydrogenases (IDHs) are key regulators of flux through biosynthetic and oxidative pathways in response to cellular energy levels. Here we present the first structures of a eukaryotic member of this enzyme family, the allosteric, hetero-octameric, NAD(+)-specific IDH from yeast in three forms: 1) without ligands, 2) with bound analog citrate, and 3) with bound citrate + AMP. The structures reveal the molecular basis for ligand binding to homologous but distinct regulatory and catalytic sites positioned at the interfaces between IDH1 and IDH2 subunits and define pathways of communication between heterodimers and heterotetramers in the hetero-octamer. Disulfide bonds observed at the heterotetrameric interfaces in the unliganded IDH hetero-octamer are reduced in the ligand-bound forms, suggesting a redox regulatory mechanism that may be analogous to the "on-off" regulation of non-allosteric bacterial IDHs via phosphorylation. The results strongly suggest that eukaryotic IDH enzymes are exquisitely tuned to ensure that allosteric activation occurs only when concentrations of isocitrate are elevated.

Highlights

The oxidative decarboxylation of isocitrate to ␣-ketoglutarate catalyzed by mitochondrial NADϩ-specific isocitrate dehydrogenases is a rate-limiting step in the tricarboxylic acid cycle
Yeast IDH is a hetero-octamer composed of four IDH1 (Mr ϭ 38,001) and four IDH2 (Mr ϭ 37,755) subunits with 42% sequence identity (6 – 8)
Mammalian NADϩ-specific isocitrate dehydrogenases are structurally more complex octamers than yeast IDH, containing three subunits in a ratio of ␣2:␤:␥, with the ␣-subunit being more similar in sequence to the catalytic yeast IDH2, and the ␤- and ␥-subunits apparently contributing regulatory functions similar to those provided by IDH1 [15,16,17,18,19]

Summary

Introduction

The oxidative decarboxylation of isocitrate to ␣-ketoglutarate catalyzed by mitochondrial NADϩ-specific isocitrate dehydrogenases is a rate-limiting step in the tricarboxylic acid cycle. The structures reveal the molecular basis for ligand binding to homologous but distinct regulatory and catalytic sites positioned at the interfaces between IDH1 and IDH2 subunits and define pathways of communication between heterodimers and heterotetramers in the hetero-octamer.

Results

Conclusion