Ser95, Asn97, and Thr78 are important for the catalytic function of porcine NADP‐dependent isocitrate dehydrogenase

Kim, Tae Kang; Colman, Roberta F.

doi:10.1110/ps.041091805

Cited by 8 publications

(4 citation statements)

References 28 publications

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“…The T77A and S94A mutants possess about 26% and 22% activity, whereas their K m values are increased by about 15-and 10-fold, respectively; and the double mutation shows a synergetic effect ( Table 2), indicating that these two residues play more important roles in ICT binding than in catalysis. These results are consistent with the biochemical studies of the equivalent residues (Thr78, Ser95, and Arg101) of PmIDH [24,26]. Considering that the K m value can be affected by other factors such as the rate of conversion of the bound substrate to the product and hence may not accurately reflect the binding affinity between the substrate and the enzyme, we further analyzed the thermodynamics of ICT binding by the wild-type and mutant IDH1 using isothermal titration calorimetry (ITC; Figure 3).…”

Section: Site I Is An Initial Ict-binding Sitesupporting

confidence: 91%

“…As shown in Table 2, the V max and K m (ICT) values of the wild-type IDH1 are comparable to that of PmIDH and E. coli NADP-IDH (EcIDH) [22][23][24][25][26]. Compared with the wild-type IDH1, the homodimeric R132H mutant manifests a 200-fold increase in the K m whereas its specific activity is decreased to about 5%; similarly, the K m of the R132A mutant is also substantially increased by 106-fold while its specific activity is decreased to about 18% ( Table 2).…”

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

confidence: 66%

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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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Section: Site I Is An Initial Ict-binding Sitesupporting

confidence: 91%

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

confidence: 66%

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

et al. 2010

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“…Despite some differences in pH optima and pI values, the kinetic properties of the mouse IDP1 and IDP2 enzymes are quite similar. The mammalian IDP1 enzyme has been extensively characterized [22,43,44], and our values for the mouse enzyme are similar to those reported for the purified recombinant human enzyme [44]. In contrast, although the crystal structures of both mamIDP1 [45] and mamIDP2 [46] have been reported, there have been fewer kinetic studies of the mamIDP2 enzyme.…”

Section: Discussionsupporting

confidence: 79%

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

Minard

McAlister-Henn

2008

Archives of Biochemistry and Biophysics

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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. Mouse IDP2 was found to similarly colocalize in both cellular compartments when expressed in yeast at levels equivalent to those of endogenous yeast isozymes. The mouse enzyme can compensate for loss of yeast cytosolic IDP2 and of peroxisomal IDP3. Removal of the peroxisomal targeting signal of the mouse enzyme precludes both localization in peroxisomes and compensation for loss of yeast IDP3.

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“…IDH has a critical metabolic function and consequently is found in organisms from all domains of life. This enzyme has been extensively studied both kinetically and structurally from psychrophilic, mesophilic and (hyper)thermophilic organisms [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. With respect to cofactor specificity and oligometric states the IDH enzyme represents a diverse family which is divided into three subfamilies based on the primary sequences [15].…”

Section: Introductionmentioning

confidence: 99%

The complex structures of isocitrate dehydrogenase from Clostridium thermocellum and Desulfotalea psychrophila suggest a new active site locking mechanism

et al. 2012

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Isocitrate dehydrogenase (IDH) catalyzes the oxidative NAD(P)+‐dependent decarboxylation of isocitrate into α‐ketoglutarate and CO2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH (CtIDH), a native open apo CtIDH to 2.35 Å and a quaternary complex of CtIDH with NADP+, isocitrate and Mg2+ to 2.5 Å. To compare to these a quaternary complex structure of the psychrophilic Desulfotalea psychrophila IDH (DpIDH) was also resolved to 1.93 Å. CtIDH and DpIDH showed similar global thermal stabilities with melting temperatures of 67.9 and 66.9 °C, respectively. CtIDH represents a typical thermophilic enzyme, with a large number of ionic interactions and hydrogen bonds per residue combined with stabilization of the N and C termini. CtIDH had a higher activity temperature optimum, and showed greater affinity for the substrates with an active site that was less thermolabile compared to DpIDH. The uncompensated negative surface charge and the enlarged methionine cluster in the hinge region both of which are important for cold activity in DpIDH, were absent in CtIDH. These structural comparisons revealed that prokaryotic IDHs in subfamily II have a unique locking mechanism involving Arg310, Asp251′ and Arg255 (CtIDH). These interactions lock the large domain to the small domain and direct NADP+ into the correct orientation, which together are important for NADP+ selectivity.

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Ser⁹⁵, Asn⁹⁷, and Thr⁷⁸ are important for the catalytic function of porcine NADP‐dependent isocitrate dehydrogenase

Cited by 8 publications

References 28 publications

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

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

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

The complex structures of isocitrate dehydrogenase from Clostridium thermocellum and Desulfotalea psychrophila suggest a new active site locking mechanism

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