Ligand binding and structural changes associated with allostery in yeast NAD(+)-specific isocitrate dehydrogenase.

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four each of regulatory IDH1 and catalytic IDH2 subunits that share 42% sequence identity. IDH2 contains catalytic isocitrate/Mg2+ and NAD+ binding sites whereas IDH1 contains homologous binding sites, respectively, for cooperative binding of isocitrate and for allosteric binding of AMP. Ligand binding is highly ordered in vitro, and IDH exhibits the unusual property of half-site binding for all ligands.

Basis for half-site ligand binding in yeast NAD(+)-specific isocitrate dehydrogenase.

Yeast NAD(+)-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four heterodimers of a catalytic IDH2 subunit and a regulatory IDH1 subunit. Despite structural predictions that the enzyme would contain eight isocitrate binding sites, four NAD(+) binding sites, and four AMP binding sites, only half of the sites for each ligand can be measured in binding assays. On the basis of a potential interaction between side chains of Cys-150 residues in IDH2 subunits in each tetramer of the enzyme, ligand binding assays of wild-type (IDH1/IDH2) and IDH1/IDH2(C150S) octameric enzymes were conducted in the presence of dithiothreitol.

Effects of excess succinate and retrograde control of metabolite accumulation in yeast tricarboxylic cycle mutants.

Cellular and mitochondrial metabolite levels were measured in yeast TCA cycle mutants (sdh2Δ or fum1Δ) lacking succinate dehydrogenase or fumarase activities. Cellular levels of succinate relative to parental strain levels were found to be elevated ~8-fold in the sdh2Δ mutant and ~4-fold in the fum1Δ mutant, and there was a preferential increase in mitochondrial levels in these mutant strains. The sdh2Δ and fum1Δ strains also exhibited 3-4-fold increases in expression of Cit2, the cytosolic form of citrate synthase that functions in the glyoxylate pathway.

Construction and analyses of tetrameric forms of yeast NAD+-specific isocitrate dehydrogenase.

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four heterodimers of regulatory IDH1 and catalytic IDH2 subunits. The crystal structure suggested that the interactions between tetramers in the octamer are restricted to defined regions in IDH1 subunits from each tetramer. Using truncation and mutagenesis, we constructed three tetrameric forms of IDH.

Pnc1p supports increases in cellular NAD(H) levels in response to internal or external oxidative stress.

Following transfer from medium with fermentable glucose to medium with nonfermentable acetate as the carbon source, cellular levels of NAD(H) were found to increase approximately 2-fold in a parental yeast strain. Similar transfer of a mutant strain subject to endogenous oxidative stress under these conditions produced more dramatic increases in cellular levels of NAD(H), and elevations above parental levels were shown to be due to the nicotinimidase Pnc1p. Similar transient increases in NAD(H) levels observed in the parental strain following addition of exogenous hydrogen peroxide were also attributable to Pnc1p.

Peroxisomal localization and function of NADP+ -specific isocitrate dehydrogenases in yeast.

Yeast peroxisomal NADP(+)-specific isocitrate dehydrogenase (IDP3) contains a canonical type I peroxisomal targeting sequence (a carboxyl-terminal Cys-Lys-Leu tripeptide), and provides the NADPH required for beta-oxidation of some fatty acids in that organelle. Cytosolic yeast IDP2 carrying a PTS1 (IDP2(+CKL)) was only partially localized to peroxisomes, and the enzyme was able to function in lieu of either peroxisomal IDP3 or cytosolic IDP2. The analogous isocitrate dehydrogenase enzyme (IDPA) from Aspergillus nidulans, irrespective of the presence or absence of a putative PTS1, was found to exhibit patterns of dual compartmental distribution and of dual function in yeast similar to those observed for IDP2(+CKL).

Disulfide bond formation in yeast NAD+-specific isocitrate dehydrogenase.

The tricarboxylic acid cycle NAD+-specific isocitrate dehydrogenase (IDH) of Saccharomyces cerevisiae is an octameric enzyme composed of four heterodimers of regulatory IDH1 and catalytic IDH2 subunits. Recent structural analyses revealed the close proximity of Cys-150 residues from IDH2 in adjacent heterodimers, and features of the structure for the ligand-free enzyme suggested that formation of a disulfide bond between these residues might stabilize an inactive form of the enzyme. We constructed two mutant forms of IDH, one containing a C150S substitution in IDH2 and the other containing C56S/C242S substitutions in IDH2 leaving Cys-150 as the sole cysteine residue.

Suppression of metabolic defects of yeast isocitrate dehydrogenase and aconitase mutants by loss of citrate synthase.

Yeast mutants lacking mitochondrial NAD(+)-specific isocitrate dehydrogenase (idhDelta) or aconitase (aco1Delta) were found to share several growth phenotypes as well as patterns of specific protein expression that differed from the parental strain. These shared properties of idhDelta and aco1Delta strains were eliminated or moderated by co-disruption of the CIT1 gene encoding mitochondrial citrate synthase. Gas chromatography/mass spectrometry analyses indicated a particularly dramatic increase in cellular citrate levels in idhDelta and aco1Delta strains, whereas citrate levels were substantially lower in idhDeltacit1Delta and aco1Deltacit1Delta strains.

Dual compartmental localization and function of mammalian NADP+-specific isocitrate dehydrogenase in yeast.

Isozymes of NADP+-specific isocitrate dehydrogenase (IDP) provide NADPH in cytosolic, mitochondrial, and peroxisomal compartments of eukaryotic cells. Analyses of purified IDP isozymes from yeast and from mouse suggest a general correspondence of pH optima for catalysis and pI values with pH values reported for resident cellular compartments. However, mouse IDP2, which partitions between cytosolic and peroxisomal compartments in mammalian cells, exhibits a broad pH optimum and an intermediate pI value.

Allosteric motions in structures of yeast NAD+-specific isocitrate dehydrogenase.

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.

Crystal structure of the yeast nicotinamidase Pnc1p.

The yeast nicotinamidase Pnc1p acts in transcriptional silencing by reducing levels of nicotinamide, an inhibitor of the histone deacetylase Sir2p. The Pnc1p structure was determined at 2.9A resolution using MAD and MIRAS phasing methods after inadvertent crystallization during the pursuit of the structure of histidine-tagged yeast isocitrate dehydrogenase (IDH).

Novel allosteric properties produced by residue substitutions in the subunit interface of yeast NAD+-specific isocitrate dehydrogenase.

Yeast NAD+-specific isocitrate dehydrogenase (IDH) is an octamer of four IDH1 and four IDH2 subunits, and the basic structural unit of the enzyme is an IDH1/IDH2 heterodimer. To investigate one aspect of the interaction between IDH1 and IDH2, residues in a hydrophobic region at the heterodimer interface (Val-216, Ser-220, and Val-224 in IDH1; Ile-221, Val-225, and Val-229 in IDH2) were replaced by alanine residues in each and in both subunits. Gel filtration and sedimentation velocity analyses demonstrated that the residue substitutions do not disrupt the octameric structure of IDH.

Physiological consequences of loss of allosteric activation of yeast NAD+-specific isocitrate dehydrogenase.

Based on allosteric regulatory properties, NAD+-specific isocitrate dehydrogenase (IDH) is believed to control flux through the tricarboxylic acid cycle in vivo. To distinguish growth phenotypes associated with regulatory dysfunction of this enzyme in Saccharomyces cerevisiae, we analyzed strains expressing well defined mutant forms of IDH or a non-allosteric bacterial NAD+-specific isocitrate dehydrogenase (IDHa). As previously reported, expression of mutant forms of IDH with severe catalytic defects but intact regulatory properties produced an inability to grow with acetate as the carbon source and a dramatic increase in the frequency of generation of petite colonies, phenotypes also exhibited by a strain (idh1Deltaidh2Delta) lacking IDH.

Crystallization and preliminary X-ray crystallographic analysis of yeast NAD+-specific isocitrate dehydrogenase.

NAD+-specific isocitrate dehydrogenase (IDH; EC 1.1.1.

Analysis of interactions with mitochondrial mRNA using mutant forms of yeast NAD(+)-specific isocitrate dehydrogenase.

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an allosterically regulated tricarboxylic acid cycle enzyme that has been shown to bind specifically and with high affinity to 5'-untranslated regions of yeast mitochondrial mRNAs. The absence of IDH has been shown to result in reduced expression of mitochondrial translation products, leading to the suggestion that this macromolecular interaction may contribute to regulating rates of translation. The interaction with mitochondrial mRNAs also produces a dramatic inhibition of IDH catalytic activity that is specifically alleviated by AMP, the primary allosteric activator of IDH.

Sources of NADPH in yeast vary with carbon source.

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.

Kinetic properties and metabolic contributions of yeast mitochondrial and cytosolic NADP+-specific isocitrate dehydrogenases.

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 Km value approximately 7-fold lower than that of IDP2, whereas, with respect to alpha-ketoglutarate, IDP2 had an apparent Km value approximately 10-fold lower than that of IDP1.

Influence of compartmental localization on the function of yeast NADP+-specific isocitrate dehydrogenases.

Three differentially compartmentalized isozymes of isocitrate dehydrogenase (mitochondrial IDP1, cytosolic IDP2, and peroxisomal IDP3) in the yeast Saccharomyces cerevisiae catalyze the NADP(+)-dependent oxidative decarboxylation of isocitrate to form alpha-ketoglutarate. These enzymes are highly homologous but exhibit some significant differences in physical and kinetic properties. To examine the impact of these differences on physiological function, we exchanged promoters and altered organellar targeting information to obtain expression of IDP2 and IDP3 in mitochondria and of IDP1 and IDP3 in the cytosol.

Multiple cellular consequences of isocitrate dehydrogenase isozyme dysfunction.

To probe the functions of multiple forms of isocitrate dehydrogenase in Saccharomyces cerevisiae, mutants lacking three of the isozymes were constructed and analyzed. Results show that, while the mitochondrial NAD+-dependent enzyme, IDH (composed of Idh1p and Idh2p subunits) is not the major contributor to total isocitrate dehydrogenase activity under any growth condition, loss of IDH produces the most dramatic growth phenotypes. These include reduced growth in the absence of glutamate, as well as an increase in expression of Idp2p (the cytosolic NADP+-dependent enzyme) under some growth conditions.

Modulation of citrate metabolism alters aluminum tolerance in yeast and transgenic canola overexpressing a mitochondrial citrate synthase.

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.

Physical and genetic interactions of cytosolic malate dehydrogenase with other gluconeogenic enzymes.

A truncated form (deltanMDH2) of yeast cytosolic malate dehydrogenase (MDH2) lacking 12 residues on the amino terminus was found to be inadequate for gluconeogenic function in vivo because the mutant enzyme fails to restore growth of a Deltamdh2 strain on minimal medium with ethanol or acetate as the carbon source. The DeltanMDH2 enzyme was also previously found to be refractory to the rapid glucose-induced inactivation and degradation observed for authentic MDH2. In contrast, kinetic properties measured for purified forms of MDH2 and deltanMDH2 enzymes are very similar.

Global transcription analysis of Krebs tricarboxylic acid cycle mutants reveals an alternating pattern of gene expression and effects on hypoxic and oxidative genes.

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.

Homologous binding sites in yeast isocitrate dehydrogenase for cofactor (NAD+) and allosteric activator (AMP).

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an allosterically regulated octameric enzyme composed of two types of homologous subunits designated IDH1 and IDH2. Based on sequence comparisons and structural models, both subunits are predicted to have adenine nucleotide binding sites. This was tested by alanine replacement of residues in putative sites in each subunit.

Isocitrate binding at two functionally distinct sites in yeast NAD+-specific isocitrate dehydrogenase.

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octamer containing two types of homologous subunits. Ligand-binding analyses were conducted to examine effects of residue changes in putative catalytic and regulatory isocitrate-binding sites respectively contained in IDH2 and IDH1 subunits. Replacement of homologous serine residues in either subunit site, S98A in IDH2 or S92A in IDH1, was found to reduce by half the total number of holoenzyme isocitrate-binding sites, confirming a correlation between detrimental effects on isocitrate binding and respective kinetic defects in catalysis and allosteric activation by AMP.