Kinetic Properties and Metabolic Contributions of Yeast Mitochondrial and Cytosolic NADP+-specific Isocitrate Dehydrogenases

Abstract

To compare kinetic properties of homologous isozymes of NADP ؉ -specific isocitrate dehydrogenase, histidine-tagged forms of yeast mitochondrial (IDP1) and cytosolic (IDP2) enzymes were expressed and purified. The isozymes were found to share similar apparent affinities for cofactors. However, with respect to isocitrate, IDP1 had an apparent K m value ϳ7-fold lower than that of IDP2, whereas, with respect to ␣-ketoglutarate, IDP2 had an apparent K m value ϳ10-fold lower than that of IDP1. Similar K m values for substrates and cofactors in decarboxylation and carboxylation reactions were obtained for IDP2, suggesting a capacity for bidirectional catalysis in vivo. Concentrations of isocitrate and ␣-ketoglutarate measured in extracts from the parental strain were found to be similar with growth on different carbon sources. For mutant strains lacking IDP1, IDP2, and/or the mitochondrial NAD ؉ -specific isocitrate dehydrogenase (IDH), metabolite measurements indicated that major cellular flux is through the IDH-catalyzed reaction in glucose-grown cells and through the IDP2-catalyzed reaction in cells grown with a nonfermentable carbon source (glycerol and lactate). A substantial cellular pool of ␣-ketoglutarate is attributed to IDH function during glucose growth, and to both IDP1 and IDH function during growth on glycerol/lactate. Complementation experiments using a strain lacking IDH demonstrated that overexpression of IDP1 partially compensated for the glutamate auxotrophy associated with loss of IDH. Collectively, these results suggest an ancillary role for IDP1 in cellular glutamate synthesis and a role for IDP2 in equilibrating and maintaining cellular levels of isocitrate and ␣-ketoglutarate.The existence of multiple and differentially compartmentalized isozymes of NADP ϩ -specific isocitrate dehydrogenase (IDP) 1 appears to be a common attribute of eukaroytic organisms. Whereas Saccharomyces cerevisiae has three isozymes encoded by different genes (1-4), the cytosolic and peroxisomal isozymes of mammalian cells are encoded by a single gene distinct from that encoding the mitochondrial isozyme (5-7). In Arabidopsis, genes for cytoplasmic and peroxisomal isozymes are distinct, but a third gene apparently encodes both mitochondrial and chloroplast isozymes (8). This duplication of genes and/or differential localization of the same enzyme suggests that the reaction catalyzed by IDP isozymes is important for optimal metabolic function of various cellular compartments. The current study is directed toward refining our understanding of these metabolic functions.The yeast mitochondrial IDP1, cytosolic IDP2, and peroxisomal IDP3 isozymes are homodimers, and they share pairwise primary sequence identities of Ͼ70% (1-4). They differ with respect to organellar targeting sequences; IDP1 has a 16-residue amino-terminal sequence that is removed upon mitochondrial import (1), and IDP3 has a carboxyl-terminal Cys-LysLeu tripeptide essential for peroxisomal localization (3, 4). IDP2 also has a significantly lower isoelectric p...

Increased superoxide formation induced by irradiation preconditioning triggers kidney resistance to ischemia-reperfusion injury in mice

“…Han et al (9) reported that sublethal whole-body irradiation marginally increased the expression of MnSOD mRNA and protein in mice spleens but significantly decreased catalase and CuZnSOD activity. In this study, the authors suggested that MnSOD played an important role in radiation-induced cellular damage and that the increase in MnSOD activity may be used as a rapid predictive assay of radiosensitivity in the clinical setting (31).…”

The 2‐oxoglutarate supply exerts significant control on the lysine synthesis flux in Saccharomyces cerevisiae

“…3 and Table 3). Lack of one of the two isoforms of the NAD + -dependent isocitrate dehydrogenase in the idh2D strain, and lack of one of the three isoforms of the NADP + -dependent isocitrate dehydrogenase in the idp1D strain, produced significantly lower 2-OG contents [32,33] (Fig. 3 and Table 3).…”

“…For 2-OG extraction and intracellular content determination, 100 mL of cell culture at an attenuance at 600 nm of 2.0 were harvested by rapid centrifugation and extracted in boiling water as previously described [33]. Determination of Lys incorporated into proteins (Lys prot ) was performed as follows.…”

Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

“…15) However, in A. niger, the alterations in the specific activity of NADP + -ICDH, but not NAD + -ICDH, are related to the extremely high production of citric acid. 25,[27][28][29] In addition, neither the gene expression nor the enzymatic activity of NAD + -ICDH are affected by the increases in the transcript level of the NADP + -ICDH gene and the specific activity of its product.…”

“…Idp1p contains mitochondrial targeting signal peptides in its N-terminus, 11) Idp3p contains peroxisomal targeting signal peptides in its C-terminus, 12,13) and Idp2p contains no targeting signal peptides and is localized in the cytoplasm. 14) Because intracellular concentrations of citric acid and α-ketoglutaric acid in idp2 disruptants are markedly lower than those in idp1 disruptants, 15) mitochondrial NADP + -ICDH (Idp1p) may play a minor role in supplying α-ketoglutaric acid via the TCA cycle compared to mitochondrial NAD + -ICDH. Codisruption of idp2 in idh2 disruptants results in glutamate auxotrophy, indicating that cytosolic NADP + -ICDH (Idp2p) contributes to the synthesis of glutamic acid.…”