Single Arginine Mutation in Two Yeast Isocitrate Dehydrogenases: Biochemical Characterization and Functional Implication

Song, Ping; Wei, Huanhuan; Cao, Zhengyu; Wang, Peng; Zhu, Guoping

doi:10.1371/journal.pone.0115025

Cited by 7 publications

(4 citation statements)

References 30 publications

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“…The kinetic data are summarized in Table , including EcIDH and its R153 mutants, and human IDH1 and its R132 mutants from different reports. The catalytic turnover numbers ( k cat ) toward isocitrate of wild‐type EcIDH (51.8 s −1 ) is very close to the result reported by Zhao et al (56.9 s −1 ) , which is comparable to that of wild‐type human IDH1 reported by Song et al (15.6 s −1 ) , Pietrak et al (12.5 s −1 ) and Yang et al (11 s −1 ) . The huge discrepancies in the kinetic properties determined for wild‐type human IDH1 by different other groups, for example, the k cat for isocitrate reported by Gross et al was >10,000‐fold larger than that reported by Yang et al, are perplexing .…”

Section: Resultssupporting

confidence: 83%

“…Interestingly, series of phenotypic changes caused by IDH mutation could be abolished by coexpression of a homolog of D‐2‐hydroxyglutaric acid dehydrogenase in Drosophila . Single arginine mutation in two yeast mitochondrial IDHs, Arg148 of Saccharomyces cerevisiae NADP + ‐IDH1 (ScIDH1) and Arg141 of Yarrowia lipolytica NADP + ‐IDH (YlIDH), caused similar effects that both ScIDH1 R148H and YlIDH R141H acquired the neomorphic activity of catalyzing a‐KG to 2‐HG . It is interesting that the analogous R153 mutants of EcIDH displayed similar catalytic properties, which is the first case in prokaryote to show the replacement effect of Arg with His.…”

Section: Discussionmentioning

confidence: 99%

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Point mutation (R153H or R153C) in Escherichia coli isocitrate dehydrogenase: Biochemical characterization and functional implication

Song

et al. 2016

J. Basic Microbiol.

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Arginine 132 (R132) mutations to histidine or cysteine frequently occur to cytosolic NADP -isocitrate dehydrogenase (IDH1) in secondary glioblastoma multiforme (GBM) patients, in which GBM develops from a lower grade astroctyoma. Mutant enzymes lose the normal IDH activity, but acquire a neomorphic ability of producing 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG). In the present study, the analogous mutations, Arg to His or Cys, were employed to homologous Arg153 of the NADP -IDH from Escherichia coli (EcIDH), generating two mutants: EcIDH R153 H and EcIDH R153C. The mutations dramatically reduced the catalytic efficiencies (k /K ) of EcIDH R153H and EcIDH R153C for isocitrate oxidation, which dropped to only 0.6 and 1.5% of the wild-type enzyme, respectively. Neoenzymatic activities of catalyzing α-KG to 2-HG by EcIDH R153H and EcIDH R153C were confirmed by GC/TOF-MS analysis. The K values of EcIDH R153H and EcIDH R153C displayed for α-KG were 3.3 ± 0.12 and 2.2 ± 0.13 mM, respectively, and the catalytic efficiencies (k /K ) of the two mutants for α-KG were 300 and 450 M s , respectively. As human IDH1 Arg132 mutation is cancer-associated, the present study provides new information for the in-depth investigation of the metabolic influence of EcIDH Arg mutation in vivo.

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Section: Resultssupporting

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Point mutation (R153H or R153C) in Escherichia coli isocitrate dehydrogenase: Biochemical characterization and functional implication

Song

et al. 2016

J. Basic Microbiol.

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“…Site-directed substitutions of protein amino acids may impact organismal fitness by different mechanisms, in addition to the loss of a specific function (Tokuriki and Tawfik 2009;Jeffery 2011;Song et al 2014). Therefore, we inspected if particular amino acid replacements in the catalytic mutants accounted for dominant effects on fitness.…”

Section: Selection Of Enzyme-coding Genes and Catalytic Mutant Designmentioning

confidence: 99%

Protein Moonlighting Revealed by Noncatalytic Phenotypes of Yeast Enzymes

et al. 2018

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A single gene can partake in several biological processes, and therefore gene deletions can lead to different-sometimes unexpected-phenotypes. However, it is not always clear whether such pleiotropy reflects the loss of a unique molecular activity involved in different processes or the loss of a multifunctional protein. Here, using metabolism as a model, we systematically test the null hypothesis that enzyme phenotypes depend on a single annotated molecular function, namely their catalysis. We screened a set of carefully selected genes by quantifying the contribution of catalysis to gene deletion phenotypes under different environmental conditions. While most phenotypes were explained by loss of catalysis, slow growth was readily rescued by a catalytically inactive protein in about one-third of the enzymes tested. Such noncatalytic phenotypes were frequent in the Alt1 and Bat2 transaminases and in the isoleucine/valine biosynthetic enzymes Ilv1 and Ilv2, suggesting novel "moonlighting" activities in these proteins. Furthermore, differential genetic interaction profiles of gene deletion and catalytic mutants indicated that is functionally associated with regulatory processes, specifically to chromatin modification. Our systematic study shows that gene loss phenotypes and their genetic interactions are frequently not driven by the loss of an annotated catalytic function, underscoring the moonlighting nature of cellular metabolism.

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“…But even though binding sites are very different, all of these mutants, with the exception of IDH1 R100Q, produce 2HG. Even more, it has been reported that mutating IDH1 R132H analogous positions in isocitrate dehydrogenase enzymes leads to 2HG production in at least two yeast species (Song et al, 2014).…”

Section: The Binding Sites Of the Mutant Enzymes Are Different Among mentioning

confidence: 99%

IDH1 and IDH2 mutants identified in cancer lose inhibition by isocitrate because of a change in their binding sites

Bascur

Alegría-Arcos

Araya-Durán

et al. 2018

Preprint

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IDH1 and IDH2 are human enzymes that convert isocitrate (ICT) into α-ketoglutarate (AKG). However, mutations in positions R132 of IDH1 and R140 and R172 of IDH2 cause these enzymes to convert AKG into 2-hydroxyglutarate (2HG). Concurrently, accumulation of 2HG in the cell is correlated with the development of cancer. This activity change is mainly due to the loss of the competitive inhibition by ICT of these enzymes, but the molecular mechanism behind this loss of inhibition is currently unknown. In this work we characterized the inhibition and loss of inhibition of IDH1 and IDH2 by means of the binding energies derived from molecular docking calculations. We characterized the substrate binding sites and how they differ among the mutant and wild type enzymes using a Jaccard similarity coefficient based on the residues involved in binding the substrates. We found that molecular docking effectively identifies the inhibition by ICT in the wild type and mutant enzymes that do not appear in tumors, and the loss of inhibition in the mutant enzymes that appear in tumors. Additionally, we found that the binding sites of the mutant enzymes are different among themselves. Finally, we found that the regulatory segment of IDH1 plays a prominent role in the change of binding sites between the mutant enzymes and the wildtype enzymes. Our findings show that the loss of inhibition is related to variations in the enzyme binding sites. Additionally, our findings show that a drug capable of targeting all IDH1 and IDH2 mutations in cancer is unlikely to be found due to significant differences among the binding sites of these paralogs. Moreover, the methodology developed here, which combines molecular docking calculations with binding site similarity estimation, can be useful for engineering enzymes, for instance, when aiming to modify the substrate affinity of an enzyme.

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Single Arginine Mutation in Two Yeast Isocitrate Dehydrogenases: Biochemical Characterization and Functional Implication

Cited by 7 publications

References 30 publications

Point mutation (R153H or R153C) in Escherichia coli isocitrate dehydrogenase: Biochemical characterization and functional implication

Point mutation (R153H or R153C) in Escherichia coli isocitrate dehydrogenase: Biochemical characterization and functional implication

Protein Moonlighting Revealed by Noncatalytic Phenotypes of Yeast Enzymes

IDH1 and IDH2 mutants identified in cancer lose inhibition by isocitrate because of a change in their binding sites

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