Production of NADPH in Saccharomyces cerevisiae cells grown on glucose has been attributed to glucose-6-phosphate dehydrogenase (Zwf1p) and a cytosolic aldehyde dehydrogenase (Ald6p) (Grabowska, D., and Chelstowska, A. (2003) J. Biol. Chem. 278, 13984 -13988). This was based on compensation by overexpression of Ald6p for phenotypes associated with ZWF1 gene disruption and on the apparent lethality resulting from co-disruption of ZWF1 and ALD6 genes. However, we have found that a zwf1⌬ald6⌬ mutant can be constructed by mating when tetrads are dissected on plates with a nonfermentable carbon source (lactate), a condition associated with expression of another enzymatic source of NADPH, cytosolic NADP ؉ -specific isocitrate dehydrogenase (Idp2p). We demonstrated previously that a zwf1⌬idp2⌬ mutant loses viability when shifted to medium with oleate or acetate as the carbon source, apparently because of the inadequate supply of NADPH for cellular antioxidant systems. In contrast, the zwf1⌬ald6⌬ mutant grows as well as the parental strain in similar shifts. In addition, the zwf1⌬ald6⌬ mutant grows slowly but does not lose viability when shifted to culture medium with glucose as the carbon source, and the mutant resumes growth when the glucose is exhausted from the medium. Measurements of NADP(H) levels revealed that NADPH may not be rapidly utilized in the zwf1⌬ald6⌬ mutant in glucose medium, perhaps because of a reduction in fatty acid synthesis associated with loss of Ald6p. In contrast, levels of NADP ؉ rise dramatically in the zwf1⌬idp2⌬ mutant in acetate medium, suggesting a decrease in production of NADPH reducing equivalents needed both for biosynthesis and for antioxidant functions.Reducing equivalents in the form of NADPH are required for numerous biosynthetic enzymatic reactions and antioxidant mechanisms involving glutathione and/or thioredoxin. The major cellular source of NADPH is thought to be the hexose monophosphate pathway. However, disruption of the Saccharomyces cerevisiae ZWF1 gene encoding glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme in that pathway, was found to produce relatively mild growth phenotypes including methionine auxotrophy and an increased sensitivity to exogenous oxidizing agents like hydrogen peroxide (1, 2). In studies designed to identify other crucial sources of NADPH in yeast, co-disruption of ZWF1 and the gene (IDP2) encoding cytosolic NADP ϩ -specific isocitrate dehydrogenase was found to produce a rapid loss in cell viability following shifts from medium containing glucose to medium containing either oleate or acetate as the carbon source (3, 4). This loss of viability correlated with an increase in levels of endogenous cellular oxidants. Fatty acid metabolism requires rapid flux through ␤-oxidation, a peroxisomal process in yeast that produces hydrogen peroxide in the first reaction of each cycle (5, 6), and acetate is a stringent carbon source that produces rapid flux through mitochondrial respiration, a process associated with the generation of dele...

Aluminum (Al) toxicity is a major constraint for crop production in acid soils, although crop cultivars vary in their tolerance to Al. We have investigated the potential role of citrate in mediating Al tolerance in Al-sensitive yeast (Saccharomyces cerevisiae; MMYO11) and canola (Brassica napus cv Westar). Yeast disruption mutants defective in genes encoding tricarboxylic acid cycle enzymes, both upstream (citrate synthase [CS]) and downstream (aconitase [ACO] and isocitrate dehydrogenase [IDH]) of citrate, showed altered levels of Al tolerance. A triple mutant of CS (⌬cit123) showed lower levels of citrate accumulation and reduced Al tolerance, whereas ⌬aco1-and ⌬idh12-deficient mutants showed higher accumulation of citrate and increased levels of Al tolerance. Overexpression of a mitochondrial CS (CIT1) in MMYO11 resulted in a 2-to 3-fold increase in citrate levels, and the transformants showed enhanced Al tolerance. A gene for Arabidopsis mitochondrial CS was overexpressed in canola using an Agrobacterium tumefaciens-mediated system. Increased levels of CS gene expression and enhanced CS activity were observed in transgenic lines compared with the wild type. Root growth experiments revealed that transgenic lines have enhanced levels of Al tolerance. The transgenic lines showed enhanced levels of cellular shoot citrate and a 2-fold increase in citrate exudation when exposed to 150 m Al. Our work with yeast and transgenic canola clearly suggest that modulation of different enzymes involved in citrate synthesis and turnover (malate dehydrogenase, CS, ACO, and IDH) could be considered as potential targets of gene manipulation to understand the role of citrate metabolism in mediating Al tolerance.Aluminum toxicity is one of the major factors limiting crop productivity in acid soils. The root apex is considered the primary site of Al-induced injury, and inhibition of root elongation is one of the most visible symptoms of Al toxicity. Although most plants are sensitive to Al, several crop species exhibit genetic variation in their ability to tolerate Al. One possible mechanism of Al tolerance is the chelation of Al by organic anions within root cells or in the rhizosphere (Taylor, 1991). Exudation of a variety of low M r organic anions such as citrate, malate, or oxalate has been reported in several crop species upon exposure to Al (Miyasaka et al., 1991; Basu et al., 1994;Pellet et al., 1995;Larsen et al., 1998). Although a rapid release of organic anions (malate 2Ϫ ) was observed in near isogenic, Al-tolerant wheat (Triticum aestivum) lines (Delhaize et al., 1993), several lines of evidence suggest that a lag phase may also exist between exposure of roots to Al and excretion of organic anions. Citrate exudation from roots of rye was observed only after 120 h of exposure (Li et al., 2000). Similarly, an Alinduced, de novo synthesis of malate leading to enhanced malate efflux 24 h after exposure was demonstrated in Al-tolerant wheat cv Katepwa (Basu et al., 1994). Delayed exudation of organic anions could possibly ...

The cDNA for porcine mitochondrial NADP-specific isocitrate dehydrogenase was isolated from a lambda gt11 library using polymerase chain reaction. Translation of the DNA sequence gave a 413-residue amino acid sequence and a calculated molecular weight of 46,600 for the mature polypeptide. Previously determined peptide sequences for the amino terminus and for internal tryptic peptides were all contained within the translated sequence. The porcine protein was found to share 63% residue identity with yeast mitochondrial NADP-specific isocitrate dehydrogenase and to be immunoreactive with an antiserum against the yeast protein. Highly conserved regions include residues which have been implicated in substrate and cofactor binding in previous studies of the porcine enzyme. The two eucaryotic enzymes exhibit only minimal homology with the NADP-dependent isocitrate dehydrogenase from Escherichia coli, with the exception of a striking conservation of residues implicated in formation of the metal-isocitrate site of the procaryotic enzyme.

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The cytosolic isozyme of NADP-specific isocitrate dehydrogenase (IDP2) was purified from a Saccharomyces cerevisiae mutant containing a chromosomal disruption in the gene encoding the mitochondrial isozyme (IDP1). IDP2 was shown to be a homodimer with a subunit molecular weight of approximately 45,000 and an isoelectric point of 5.5. Amino acid sequences were obtained for tryptic peptides of IDP2 and used to plan polymerase chain reactions. A resulting 400 bp DNA fragment was used as a hybridization probe to isolate the IDP2 gene from a yeast genomic DNA library. The complete nucleotide sequence of the IDP2 coding region was determined and translated into a 412-residue amino acid sequence. IDP2 and IDP1 were found to be identical in 71% of the aligned residue positions. The identity of the IDP2 gene was confirmed by genomic replacement with a disrupted IDP2 coding region. Haploid yeast strains lacking either or both IDP2 and IDP1 were constructed by genetic crosses of mutant strains containing disruptions in chromosomal IDP2 and IDP1 loci. No dramatic differences in growth rates with common carbon sources could be attributed to these disruptions.

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.The oxidative decarboxylation of isocitrate to ␣-ketoglutarate catalyzed by mitochondrial NAD ϩ -specific isocitrate dehydrogenases is a rate-limiting step in the tricarboxylic acid cycle. The affinities of the yeast and human enzymes (IDH, 5 EC 1.1.1.41) for isocitrate are allosterically regulated positively by AMP and ADP, respectively, and the human enzyme is also regulated negatively by ATP (1, 2). Both enzymes are negatively regulated by NADH (3, 4). This control has been proposed to contribute to inverse regulation of rates of energy production by oxidative pathways and by glycolysis (5) Yeast IDH is a hetero-octamer composed of four IDH1 (M r ϭ 38,001) and four IDH2 (M r ϭ 37,755) subunits with 42% sequence identity (6 -8) (supplemental Fig. 1). Results of targeted mutagenesis studies (9 -13) suggest that IDH2 contains a catalytic isocitrate/Mg 2ϩ -and NAD ϩ -binding site, whereas the homologous site in IDH1 functions in cooperative binding of isocitrate and in binding of the allosteric activator AMP. Yeast two-hybrid assays and other mutagenesis studies (13, 14) strongly suggest that a heterodimer of IDH1 and IDH2 is the fundamental structural building block of the enzyme and that IDH1 contributes a few residues to catalytic sites in IDH2, while the analogous residues of IDH2 function in the regulatory ligand-binding sites in IDH1. Visualization of subunit interactions within and between heterodimers in the holoenzyme is essential to define the molecular basis for allosteric communication between such sites and to provide information prerequisite for an understanding of the evolution of the highly regulated allosteric IDH hetero-octamer from the more ancient nonallosteric, homodimeric bacterial IDH enzymes. Mammalian NAD ϩ -specific isocitrate dehydrogenases are structurally more complex octamers than yeast IDH, containing three subunits in a ratio of ␣ 2 :␤:␥, with the ␣-s...

To understand the many roles of the Krebs tricarboxylic acid (TCA) cycle in cell function, we used DNA microarrays to examine gene expression in response to TCA cycle dysfunction. mRNA was analyzed from yeast strains harboring defects in each of 15 genes that encode subunits of the eight TCA cycle enzymes. The expression of >400 genes changed at least threefold in response to TCA cycle dysfunction. Many genes displayed a common response to TCA cycle dysfunction indicative of a shift away from oxidative metabolism. Another set of genes displayed a pairwise, alternating pattern of expression in response to contiguous TCA cycle enzyme defects: expression was elevated in aconitase and isocitrate dehydrogenase mutants, diminished in α-ketoglutarate dehydrogenase and succinyl-CoA ligase mutants, elevated again in succinate dehydrogenase and fumarase mutants, and diminished again in malate dehydrogenase and citrate synthase mutants. This pattern correlated with previously defined TCA cycle growth–enhancing mutations and suggested a novel metabolic signaling pathway monitoring TCA cycle function. Expression of hypoxic/anaerobic genes was elevated in α-ketoglutarate dehydrogenase mutants, whereas expression of oxidative genes was diminished, consistent with a heme signaling defect caused by inadequate levels of the heme precursor, succinyl-CoA. These studies have revealed extensive responses to changes in TCA cycle function and have uncovered new and unexpected metabolic networks that are wired into the TCA cycle.

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is an allosterically regulated enzyme that exists as an octamer composed of two nonidentical subunits, designated IDH1 and IDH2. To determine the contribution of each subunit to regulation and catalysis, a conserved serine residue at the proposed active site of each subunit was mutated to alanine. This mutation in IDH1 resulted in a 6-fold decrease in Vmax and a decrease in cooperativity, but little change in S0.5 for isocitrate. The mutant IDH2, in contrast, exhibited a 60-fold decrease in maximal velocity and a 2-fold reduction in S0.5 for isocitrate, but the cooperativity was unaffected. Responses to the allosteric modifier AMP also differed for the two mutant enzymes. The IDH1 mutant enzyme was not activated by AMP, whereas the IDH2 mutant enzyme exhibited an increase in isocitrate affinity in the presence of AMP similar to that observed with the wild-type enzyme. On the basis of these kinetic results, a model is presented which proposes that IDH1 functions as a regulatory subunit while IDH2 functions in catalysis. To determine if IDH1 or IDH2 alone is catalytically active, we also expressed the individual subunits in yeast strains in which the gene encoding the other subunit had been disrupted. Mitochondrial extracts from strains overexpressing solely IDH1 or IDH2 contained no detectable activity in the presence or absence of AMP. Gel filtration of these extracts showed that both IDH1 and IDH2 behaved as monomers, suggesting that the major subunit interactions within the octamer are between IDH1 and IDH2.