Mutational analysis of the catalytic residues lysine 230 and tyrosine 160 in the NADP+-dependent isocitrate dehydrogenase from Escherichia coli

Me, Lee; Dh, Dyer; Klein, Ophir D.; Jm, Bolduc; Bl, Stoddard; De, Koshland

doi:10.1021/bi00001a046

Cited by 50 publications

(49 citation statements)

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“…In the E. coli IDH structure, the analogous residue is Tyr-160, the side chain hydroxyl group of which makes a hydrogen bond to one of the substrate carboxylate groups and to the guanidinium nitrogen of Arg-153, the arginine residue equivalent to Arg-135 in IDH2. The ability to form these hydrogen bonds has been shown to be critical for proper substrate binding, as a Y160F mutant is inactive (36). It should be noted that citrate is not bound in the catalytic site in these structures.…”

Section: Resultsmentioning

confidence: 95%

Allosteric Motions in Structures of Yeast NAD+-specific Isocitrate Dehydrogenase

Taylor

Hart

et al. 2008

Journal of Biological Chemistry

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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...

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Section: Resultsmentioning

confidence: 95%

Allosteric Motions in Structures of Yeast NAD+-specific Isocitrate Dehydrogenase

Taylor

Hart

et al. 2008

Journal of Biological Chemistry

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“…The phenolic side chain of Tyr125 occupies a position totally different from those of the corresponding Tyr residues in TfIPMDH and EcICDH. Nevertheless, the Tyr125Ala mutation caused a substantial decrease in the k cat value, as do corresponding mutations in EcICDH (Tyr160) and TtIPMDH (Tyr140) (11,15,21). This result suggests that Tyr125 is involved in the catalytic function of TtHICDH.…”

Section: Discussionmentioning

confidence: 91%

Crystal Structure of Tetrameric Homoisocitrate Dehydrogenase from an Extreme Thermophile, Thermus thermophilus : Involvement of Hydrophobic Dimer-Dimer Interaction in Extremely High Thermotolerance

et al. 2005

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The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus thermophilus (TtHICDH) was determined at 1.85-Å resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main-and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related ␤-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH.Homoisocitrate dehydrogenase (HICDH) is the third enzyme involved in lysine biosynthesis through ␣-aminoadipate (19) and is a member of a family of ␤-decarboxylating dehydrogenases that includes isocitrate dehydrogenase (ICDH) in the tricarboxylic acid cycle, 3-isopropylmalate dehydrogenase (IPMDH) in leucine biosynthesis, and tartrate dehydrogenase in vitamin production (2). It has been suggested that these enzymes diverged from a common ancestral ␤-decarboxylating dehydrogenase (4, 9, 34).We have shown that in an extremely thermophilic bacterium, Thermus thermophilus HB27, lysine is synthesized through ␣-aminoadipate, although lysine is synthesized through diaminopimelate in most bacteria (13,18,20,22,23,31). We have previously characterized HICDH from T. thermophilus HB27 (TtHICDH) in detail and have shown that the enzyme has four unique features (19). The first is its substrate specificity. Although TtHICDH is essential for lysine biosynthesis in the bacterium, TtHICDH can catalyze the reaction with isocitrate as a substrate; isocitrate is the substrate of ICDH at a 20-fold-higher efficiency than toward the native substrate, homoisocitrate. This feature contrasts with HICDH from Saccharomyces cerevisiae, which utilizes only homoisocitrate as a substrate (19). Moreover, the substrate specificity is easily altered by a single mutation, Arg85Val, to lose activity for isocitrate but to acquire activity for 3-isopropylmalate, a substrate of IPMDH. The second feature is the unique subunit organization. TtHICDH is a homotetramer, while mo...

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“…There is discrepancy in the IDH activity of the R132H mutant as Zhao et al [12] showed a 2-fold decrease in the catalytic rate while Dang et al [13] reported a 1000-fold reduction, and our data are in better agreement with that reported by Zhao et al In addition, the kinetic parameters of the R132C, R132S, and R132L mutants are similar to that of the R132H mutant ( Table 2). The Y139A and K212A mutants display < 1% activity ( Table 2), indicating that Tyr139 and Lys212, like their counterparts in EcIDH and PmIDH [22,25,27], play critical roles in the catalytic reaction. Our data also show that mutations D252A and D275A have severe effects on the K m and/or k cat due to their involvements in both ICT binding and catalysis (Table 2).…”

Section: Kinetic Studies Of the Wild-type And Mutant Idh1mentioning

confidence: 99%

Molecular mechanisms of “off-on switch” of activities of human IDH1 by tumor-associated mutation R132H

et al. 2010

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Human cytosolic NADP-IDH (IDH1) has recently been found to be involved in tumorigenesis. Notably, the tumorderived IDH1 mutations identified so far mainly occur at Arg132, and mutation R132H is the most prevalent one. This mutation impairs the oxidative IDH activity of the enzyme, but renders a new reduction function of converting α-ketoglutarate (αKG) to 2-hydroxyglutarate. Here, we report the structures of the R132H mutant IDH1 with and without isocitrate (ICT) bound. The structural data together with mutagenesis and biochemical data reveal a previously undefined initial ICT-binding state and demonstrate that IDH activity requires a conformational change to a closed pre-transition state. Arg132 plays multiple functional roles in the catalytic reaction; in particular, the R132H mutation hinders the conformational changes from the initial ICT-binding state to the pre-transition state, leading to the impairment of the IDH activity. Our results describe for the first time that there is an intermediate conformation that corresponds to an initial ICT-binding state and that the R132H mutation can trap the enzyme in this conformation, therefore shedding light on the molecular mechanism of the "off switch" of the potentially tumor-suppressive IDH activity. Furthermore, we proved the necessity of Tyr139 for the gained αKG reduction activity and propose that Tyr139 may play a vital role by compensating the increased negative charge on the C2 atom of αKG during the transfer of a hydride anion from NADPH to αKG, which provides new insights into the mechanism of the "on switch" of the hypothetically oncogenic reduction activity of IDH1 by this mutation.

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Mutational analysis of the catalytic residues lysine 230 and tyrosine 160 in the NADP+-dependent isocitrate dehydrogenase from Escherichia coli

Cited by 50 publications

References 28 publications

Allosteric Motions in Structures of Yeast NAD+-specific Isocitrate Dehydrogenase

Allosteric Motions in Structures of Yeast NAD+-specific Isocitrate Dehydrogenase

Crystal Structure of Tetrameric Homoisocitrate Dehydrogenase from an Extreme Thermophile, Thermus thermophilus : Involvement of Hydrophobic Dimer-Dimer Interaction in Extremely High Thermotolerance

Molecular mechanisms of “off-on switch” of activities of human IDH1 by tumor-associated mutation R132H

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