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Structure and allosteric regulation of human NAD-dependent isocitrate dehydrogenase.

ABSTRACT: Human NAD-dependent isocitrate dehydrogenase or HsIDH3 catalyzes the decarboxylation of isocitrate into ?-ketoglutarate in the TCA cycle. HsIDH3 exists and functions as a heterooctamer composed of the ?? and ?? heterodimers, and is regulated allosterically and/or competitively by numerous metabolites including CIT, ADP, ATP, and NADH. In this work, we report the crystal structure of HsIDH3 containing a ? mutant in apo form. In the HsIDH3 structure, the ?? and ?? heterodimers form the ?₂?? heterotetramer via their clasp domains, and two ?₂?? heterotetramers form the (?₂??)₂ heterooctamer through insertion of the N-terminus of the ? subunit of one heterotetramer into the back cleft of the ? subunit of the other heterotetramer. The functional roles of the key residues at the allosteric site, the pseudo allosteric site, the heterodimer and heterodimer-heterodimer interfaces, and the N-terminal of the ? subunit are validated by mutagenesis and kinetic studies. Our structural and biochemical data together demonstrate that the allosteric site plays an important role but the pseudo allosteric site plays no role in the allosteric activation of the enzyme; the activation signal from the allosteric site is transmitted to the active sites of both ?? and ?? heterodimers via the clasp domains; and the N-terminal of the ? subunit plays a critical role in the formation of the heterooctamer to ensure the optimal activity of the enzyme. These findings reveal the molecular mechanism of the assembly and allosteric regulation of HsIDH3.

SUBMITTER: Sun P

PROVIDER: S-EPMC7752914 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Structure and allosteric regulation of human NAD-dependent isocitrate dehydrogenase.

Sun Pengkai P Liu Yan Y Ma Tengfei T Ding Jianping J

Cell discovery 20201222 1

Human NAD-dependent isocitrate dehydrogenase or HsIDH3 catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the TCA cycle. HsIDH3 exists and functions as a heterooctamer composed of the αβ and αγ heterodimers, and is regulated allosterically and/or competitively by numerous metabolites including CIT, ADP, ATP, and NADH. In this work, we report the crystal structure of HsIDH3 containing a β mutant in apo form. In the HsIDH3 structure, the αβ and αγ heterodimers form the α<sub>2</su ...[more]

PMID: 33349631

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Project description:The NAD(+)-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encoding gdhB gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from P. aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptides from Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, and Caulobacter crescentus. A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) from Saccharomyces cerevisiae or Neurospora crassa. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of gdhB is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of P. aeruginosa and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases.

Dataset Information

Structure and allosteric regulation of human NAD-dependent isocitrate dehydrogenase.

Publications

Structure and allosteric regulation of human NAD-dependent isocitrate dehydrogenase.

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