Biomedical subjects

Lee McAlister-Henn

Publications and source records attributed to Lee McAlister-Henn.

13 recordsLinked to original sources

PubMed2006

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. However, these substitutions produce novel kinetic properties including, with respect to cofactor, positive allosteric regulation by AMP and cooperativity in the absence of AMP. These allosteric properties are also apparent in NAD+-binding experiments. Despite substantial measurable activity for the mutant enzyme containing residue substitutions in both subunits, expression of this enzyme produces growth phenotypes indicative of IDH dysfunction in vivo.

Amino Acid Sequence↗

PubMed2006

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. Reduced growth rates on acetate medium were also observed with expression of enzymes with severe regulatory defects or of the bacterial IDHa enzyme, suggesting that allosteric regulation is also important for optimal growth on this carbon source. However, expression of IDHa produced no effect on petite frequency, suggesting that the intermediate petite frequencies observed for strains expressing regulatory mutant forms of IDH are likely to correlate with the slight reductions in catalytic efficiency observed for these enzymes. Finally, rates of increase in oxygen consumption were measured during culture shifts from medium with glucose to medium with ethanol as the carbon source. Strains expressing wild-type or catalytically deficient mutant forms of IDH exhibited rapid respiratory transitions, whereas strains expressing regulatory mutant forms of IDH or the bacterial IDHa enzyme exhibited much slower respiratory transitions. This suggests an important physiological role for allosteric activation of IDH during changes in environmental conditions.

Allosteric Site↗

PubMed2005

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. Using mutant forms of IDH with defined catalytic or regulatory kinetic defects, we found that residue changes altering ligand binding in the catalytic site reduce the inhibitory effect of a transcript from the mitochondrial COX2 mRNA. In contrast, residue changes altering binding of allosteric regulators do not prevent inhibition by the COX2 RNA transcript but do prevent alleviation of inhibition by AMP. Results obtained using surface plasmon resonance methods suggest that the mRNA transcript may bind at the active site of IDH. Also, the presence of AMP has little effect on overall affinity but renders the binding of mRNA ineffective in catalytic inhibition of IDH. Finally, by expressing mutant forms of IDH in vivo, we determined that detrimental effects on levels of mitochondrial translation products correlate with a substantial reduction in catalytic activity. However, concomitant loss of IDH and of citrate synthase eliminates these effects, suggesting that any role of IDH in mitochondrial translation is indirect.

Catalysis↗

PubMed2005

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. 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 zwf1Delta ald6Delta 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 zwf1Delta idp2Delta 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 zwf1Delta ald6Delta mutant grows as well as the parental strain in similar shifts. In addition, the zwf1Delta ald6Delta 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 zwf1Delta ald6Delta 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 zwf1Delta idp2Delta mutant in acetate medium, suggesting a decrease in production of NADPH reducing equivalents needed both for biosynthesis and for antioxidant functions.

Acetates↗

PubMed2005

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

NAD+-specific isocitrate dehydrogenase (IDH; EC 1.1.1.41) is a complex allosterically regulated enzyme in the tricarboxylic acid cycle. Yeast IDH is believed to be an octamer containing four catalytic IDH2 and four regulatory IDH1 subunits. Crystals of yeast IDH have been obtained and optimized using sodium citrate, a competitive inhibitor of the enzyme, as the precipitating agent. The crystals belong to space group R3, with unit-cell parameters a = 302.0, c = 112.1 A. Diffraction data were collected to 2.9 A from a native crystal and to 4.0 A using multiwavelength anomalous diffraction (MAD) methods from an osmium derivative. Initial electron-density maps reveal large solvent channels and the molecular boundaries of the allosteric IDH multimer.

Allosteric Site↗

PubMed2004

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. Similar Km 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 alpha-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 alpha-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 alpha-ketoglutarate.

Carbon↗

PubMed2004

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. Physiological function was assessed as complementation by mislocalized isozymes of defined growth defects of isocitrate dehydrogenase mutant strains. These studies revealed that the IDP isozymes are functionally interchangeable for glutamate synthesis, although mitochondrial localization has a positive impact on this function during fermentative growth. However, IDP2, whether located in mitochondria or in the cytosol, provided the highest level of defense against endogenous or exogenous oxidative stress.

Cell Compartmentation↗

PubMed2003

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. In this study, we have focused on another phenotype associated with loss of IDH, an elevated frequency of petite mutations indicating loss of functional mtDNA. Using mutant forms of IDH with altered active site residues, a correlation was observed between the high frequency of petite mutations and the loss of catalytic activity. Loss of Idp1p (the mitochondrial NADP+-dependent enzyme) and Idp2p contributes to the loss of functional mtDNA, but only in an IDH dysfunctional background. Surprisingly, overexpression of Idp1p, but not of Idp2p, was found to result in an elevated petite frequency independent of the functional state of IDH. This is the first phenotype associated with altered Idp1p. Finally, throughout this study we examined effects of loss of mitochondrial citrate synthase (Cit1p) on isocitrate dehydrogenase mutants, since defects in the CIT1 gene were previously shown to enhance growth of IDH dysfunctional strains on nonfermentable carbon sources. Loss of Cit1p was found to suppress the petite phenotype of strains lacking IDH, suggesting that these phenotypes may be linked.

DNA, Mitochondrial↗

PubMed2003

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. Yeast two-hybrid assays indicate weak interactions between MDH2 and yeast phosphoenolpyruvate carboxykinase (PCK1) and between MDH2 and fructose-1,6-bisphosphatase (FBP1). These interactions are not observed for deltanMDH2, suggesting that differences in cellular function between authentic and truncated forms of MDH2 may be related to their ability to interact with other gluconeogenic enzymes. Additional evidence was obtained for interaction of MDH2 with PCK1 using Hummel-Dreyer gel filtration chromatography, and for interactions of MDH2 with PCK1 and with FBP1 using surface plasmon resonance. Experiments with the latter technique demonstrated a much lower affinity for interaction of deltanMDH2 with PCK1 and no interaction between deltanMDH2 and FBP1. These results suggest that the interactions of MDH2 with other gluconeogenic enzymes are dependent on the amino terminus of the enzyme, and that these interactions are important for gluconeogenic function in vivo.

Amino Acid Sequence↗

PubMed2003

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. Targets included adjacent aspartate/isoleucine residues implicated as important for determining cofactor specificity in related dehydrogenases and a residue in each IDH subunit in a position occupied by histidine in other cofactor binding sites. The primary kinetic effects of D286A/I287A and of H281A replacements in IDH2 were found to be a dramatic reduction in apparent affinity of the holoenzyme for NAD(+) and a concomitant reduction in V(max). Ligand binding assays also showed that the H281A mutant enzyme fails to bind NAD(+) under conditions that are saturating for the wild-type enzyme. In contrast, the primary effect of corresponding D279A/D280A and of R274A replacements in IDH1 is a reduction in holoenzyme binding of AMP, with concomitant alterations in kinetic and isocitrate binding properties normally associated with activation by this allosteric effector. These results suggest that the nucleotide cofactor binding site is primarily contributed by the IDH2 subunit, whereas the homologous nucleotide binding site in IDH1 has evolved for regulatory binding of AMP. These results are consistent with previous studies demonstrating that the catalytic isocitrate binding sites are comprised of residues primarily contributed by IDH2, whereas sites for regulatory binding of isocitrate are contributed by analogous residues of IDH1. In this study, we also demonstrate that a prerequisite for holoenzyme binding of NAD(+) is binding of isocitrate/Mg(2+) at the IDH2 catalytic site. This is comparable to the dependence of AMP binding upon binding of isocitrate at the IDH1 regulatory site.

Adenosine Monophosphate↗

PubMed2003

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. 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 alpha-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 alpha-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.

Aconitate Hydratase↗

PubMed2003

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. A triple mutant of CS (Deltacit123) showed lower levels of citrate accumulation and reduced Al tolerance, whereas Deltaaco1- and Deltaidh12-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 micro 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↗

PubMed2002

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. Replacement of both serine residues eliminates isocitrate binding and measurable catalytic activity. The putative isocitrate-binding sites of IDH1 and IDH2 contain five identical and four nonidentical residues. Reciprocal replacement of the four nonidentical residues in either or both subunits (A108R, F136Y, T241D, and N245D in IDH1 and/or R114A, Y142F, D248T, and D252N in IDH2) was found to be permissive for isocitrate binding. This provides further evidence for two types of binding sites in IDH, although the authentic residues have been shown to be necessary for normal kinetic contributions. Finally, the mutant enzymes with residue replacements in the IDH1 site were found to be unable to bind AMP, suggesting that allosteric activation is dependent both upon binding of isocitrate at the IDH1 site and upon the changes in the enzyme normally elicited by this binding.

Adenosine Monophosphate↗