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Structural studies of Saccharomyces cerevesiae mitochondrial NADP-dependent isocitrate dehydrogenase in different enzymatic states reveal substantial conformational changes during the catalytic reaction.

ABSTRACT: Isocitrate dehydrogenases (IDHs) catalyze oxidative decarboxylation of isocitrate (ICT) into alpha-ketoglutarate (AKG). We report here the crystal structures of Saccharomyces cerevesiae mitochondrial NADP-IDH Idp1p in binary complexes with coenzyme NADP, or substrate ICT, or product AKG, and in a quaternary complex with NADPH, AKG, and Ca(2+), which represent different enzymatic states during the catalytic reaction. Analyses of these structures identify key residues involved in the binding of these ligands. Comparisons among these structures and with the previously reported structures of other NADP-IDHs reveal that eukaryotic NADP-IDHs undergo substantial conformational changes during the catalytic reaction. Binding or release of the ligands can cause significant conformational changes of the structural elements composing the active site, leading to rotation of the large domain relative to the small and clasp domains along two hinge regions (residues 118-124 and residues 284-287) while maintaining the integrity of its secondary structural elements, and thus, formation of at least three distinct overall conformations. Specifically, the enzyme adopts an open conformation when bound to NADP, a quasi-closed conformation when bound to ICT or AKG, and a fully closed conformation when bound to NADP, ICT, and Ca(2+) in the pseudo-Michaelis complex or with NADPH, AKG, and Ca(2+) in the product state. The conformational changes of eukaryotic NADP-IDHs are quite different from those of Escherichia coli NADP-IDH, for which significant conformational changes are observed only between two forms of the apo enzyme, suggesting that the catalytic mechanism of eukaryotic NADP-IDHs is more complex than that of EcIDH, and involves more fine-tuned conformational changes.

SUBMITTER: Peng Y

PROVIDER: S-EPMC2525520 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Structural studies of Saccharomyces cerevesiae mitochondrial NADP-dependent isocitrate dehydrogenase in different enzymatic states reveal substantial conformational changes during the catalytic reaction.

Peng Yingjie Y Zhong Chen C Huang Wei W Ding Jianping J

Protein science : a publication of the Protein Society 20080613 9

Isocitrate dehydrogenases (IDHs) catalyze oxidative decarboxylation of isocitrate (ICT) into alpha-ketoglutarate (AKG). We report here the crystal structures of Saccharomyces cerevesiae mitochondrial NADP-IDH Idp1p in binary complexes with coenzyme NADP, or substrate ICT, or product AKG, and in a quaternary complex with NADPH, AKG, and Ca(2+), which represent different enzymatic states during the catalytic reaction. Analyses of these structures identify key residues involved in the binding of th ...[more]

PMID: 18552125

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Project description:Cytoplasmic NADP(+)-isocitrate dehydrogenase (NADP(+)-IDH) has been purified and characterized, and its gene sequenced in many animal, plant, and yeast species. However, much less information is available on this enzyme-gene in insects. As a first step in investigating the biochemical and molecular mechanisms by which NADP(+)-IDH contributes to adaptations for flight vs. reproduction in insects, the enzyme was purified to homogeneity in the wing-dimorphic cricket, Gryllus firmus, characterized, and its corresponding gene sequenced. Using a combination of polyethylene glycol precipitation, Cibacron-Blue affinity chromatography, and hydrophobic interaction chromatography the enzyme was purified 291-fold (7% yield; specific activity = 15.8 µmol NADPH/min/mg protein). The purified enzyme exhibited a single band on SDS PAGE (46.3 kD), but consisted of two N-terminal amino acid sequences that differed in the first two amino acids. Purified enzyme exhibited standard Michaelis-Menten kinetics at pH 8.0 and 28° C (K(M(NADP+)) = 2.3 ± 0.4 µM; K(M(Na+-Isocitrate)) = 14.7 + 1.8 µM). Subunit molecular mass and K(M)S were similar to published values for NADP(+)-IDHs from a variety of vertebrate and two insect species. PCR amplification of an internal sequence using genomic DNA followed by 3' and 5' RACE yielded a nucleotide sequence of the mature protein and translated amino-acid sequences that exhibited high similarity (40-50% and 70-80%, respectively) to sequences from insect and vertebrate NADP(+)-IDHs. Two potential ATG start codons were identified. Both Nterminal amino-acid sequences matched the nucleotide sequence, consistent with both enzyme forms being transcribed from the same gene, although these variants could also be encoded by different genes. Bioinformatic analyses and differential centrifugation indicated that the majority, if not all, of the enzyme is cytoplasmic. The enzyme exhibited high specific activity in fat body, head and gut, and a single band on native PAGE.

Dataset Information

Structural studies of Saccharomyces cerevesiae mitochondrial NADP-dependent isocitrate dehydrogenase in different enzymatic states reveal substantial conformational changes during the catalytic reaction.

Publications

Structural studies of Saccharomyces cerevesiae mitochondrial NADP-dependent isocitrate dehydrogenase in different enzymatic states reveal substantial conformational changes during the catalytic reaction.

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