Functional relevance of dynamic properties of Dimeric NADP-dependent Isocitrate Dehydrogenases (original) (raw)
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Proteins: Structure, Function, and Bioinformatics, 2007
Isocitrate dehydrogenase (ICDH) is an enzyme in the Krebs cycle that catalyses the dehydrogenation and subsequent decarboxylation of isocitrate to a-ketoglutarate and CO 2 using NAD þ or NADP þ as a cofactor. 1-3 ICDH evolved early and is widely distributed among archaea, bacteria, and eukarya. Such an evolutional background is reflected in diverse primary structures, various oligomeric states, and even different cofactor specificity. 4 ICDH and isopropylmalate dehydrogenase (IPMDH) form a family of homo-dimeric decarboxylating dehydrogenases, which share a unique cofactor-binding site that differs from the Rossmann fold found in many other dehydrogenases. 5-7 Based on a wealth of structural and enzymological information accumulated on these enzymes showing a strong preference for either NAD þ or NADP þ , the family of the dehydrogenases has become a target of many design efforts to change the preference. It has been reported that multiple mutations of seven residues at the cofactor-binding site successfully switched the specificity of Ec-ICDH from a 7000-fold preference for NADP þ over NAD þ to a 200-fold preference for NAD þ over NADP þ . 8 Remarkably, the overall activity of the mutant enzyme was comparable to that of wild-type NAD þ -dependent enzymes. Structural analyses revealed the interaction between the introduced Asp-344 residue and the vicinal hydroxyl groups of the adenosine ribose moiety of NAD þ . The multiple mutations also succeeded in shifting the binding site for the adenine ring and altering the ribose ring conformation from C3 0 -endo to C2 0 -endo puckering. 8 Naturally occurring NAD þ -specific ICDH thus attracted our interest for comparative studies with engineered NAD þ -specific ICDH and NAD þ -specific IPMDHs. We chose ICDH from an aerobic chemolithotrophic bacterium, Acidithiobacillus thiooxidans, 9 for the present structural study. NAD þ -specific ICDHs have been found in some chemolithotrophic bacteria that possess a 2-oxoglutarate dehydrogenase-deficient Krebs cycle. 10,11 It has been proposed that NAD þ -specific ICDH may be reminiscent of an enzyme that functions in CO 2 fixation in an Abbreviations: ICDH, isocitrate dehydrogenase; IPMDH, isopropylmalate dehydrogenase.
Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase
Proceedings of the National Academy of Sciences, 1989
The structure of isocitrate dehydrogenase [threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42] from Escherichia coli has been solved and refined at 2.5 A resolution and is topologically different from that of any other dehydrogenase. This enzyme, a dimer of identical 416-residue subunits, is inactivated by phosphorylation at Ser-113, which lies at the edge of an interdomain pocket that also contains many residues conserved between isocitrate dehydrogenase and isopropylmalate dehydrogenase. Isocitrate dehydrogenase contains an unusual clasp-like domain in which both polypeptide chains in the dimer interlock. Based on the structure of isocitrate dehydrogenase and conservation with isopropylmalate dehydrogenase, we suggest that the active site lies in an interdomain pocket close to the phosphorylation site.
Proteins: Structure, Function, and Bioinformatics, 2006
Isocitrate and isopropylmalalte dehydrogenases are homologous enzymes important for the cell metabolism. They oxidize their substrates using NAD or NADP as cofactors. Thus, they have two specificities, towards the substrate and the cofactor, appearing in three combinations. Although many three-dimensional (3D) structures are resolved, identification of amino acids determining these specificities remains a challenge. We present computational identification and analysis of specificity-determining positions (SDPs). Besides many experimentally proven SDPs, we predict new SDPs, for example, four substrate-specific positions (103Leu, 105Thr, 337Ala, and 341Thr in IDH from E. coli) that contact the cofactor and may play a role in the recognition process. Proteins 2006;64:1001-1009. close contact (Ͻ 5 Å) with SDPs or CPs; Other, remaining residues. Contacts were calculated for IDH from E. coli, 1ai2. (C) Fraction of amino acids belonging to different prediction classes, for different functional types of residues (notation as in B).
Construction and Analyses of Tetrameric Forms of Yeast NAD+-Specific Isocitrate Dehydrogenase
Biochemistry, 2011
Yeast NAD +-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four heterodimers of regulatory IDH1 and catalytic IDH2 subunits. The crystal structure suggested that the interactions between tetramers in the octamer are restricted to defined regions in IDH1 subunits from each tetramer. Using truncation and mutagenesis, we constructed three tetrameric forms of IDH. Truncation of five residues from the amino-terminus of IDH1 did not alter the octameric form of the enzyme, but this truncation plus IDH1 G15D or IDH1 D168K residue substitutions produced tetrameric enzymes as assessed by sedimentation velocity ultracentrifugation. The IDH1 G15D substitution in the absence of any truncation of IDH1 was subsequently found to be sufficient for production of a tetrameric enzyme. The tetrameric forms of IDH exhibited ~50% reductions in V max and in cooperativity with respect to isocitrate relative to the wild-type enzyme, but they retained the property of allosteric activation by AMP. The truncated-5 IDH1/IDH2 and tetrameric enzymes were much more sensitive than the wild-type enzyme to inhibition by the oxidant diamide and concomitant formation of a disulfide bond between IDH2 Cys-150 residues. Binding of ligands reduced the sensitivity of the wild-type enzyme to diamide but had no effect on inhibition of the truncated or tetrameric enzymes. These results suggest that the octameric structure of IDH has in part evolved for regulation of disulfidebond formation and activity by ensuring the proximity of the amino terminus of an IDH1 subunit from one tetramer to the IDH2 Cys-150 residues in the other tetramer. Mitochondrial NAD +-specific isocitrate dehydrogenase catalyzes a rate-limiting step in the tricarboxylic acid cycle. The affinity of yeast isocitrate dehydrogenase (IDH)1 for isocitrate is enhanced by AMP and reduced by NADH (1,2), suggesting that rates of oxidative energy production would be increased at the level of IDH when cellular ratios of [ATP]/[AMP] and [NADH]/[NAD + ] are low and attenuated when these ratios are high. The mammalian enzyme is similarly regulated by ADP and NADH, and also negatively regulated by ATP (3,4). Yeast IDH is a hetero-octamer composed of four IDH1 and four IDH2 subunits (5). IDH1 and IDH2 have similar molecular masses of 38,001 and 37,755, respectively, and their † This work was supported by National Institutes of Health Grants GM051265 (L.M-H.) and RR022200 (B.D.). Supercomputer allocations were provided by National Science foundation TG-MCB070038 (B.D.
Applied and Environmental Microbiology, 1998
Variations of intracellular concentrations of isocitrate and NADP+ were measured throughout all growth phases of the marine bacterium Pseudomonas nautica. The intracellular isocitrate concentration tracked the intracellular protein concentration throughout all phases of growth. It rapidly increased in early exponential phase to a maximum and fell to nearly zero in parallel with pyruvate exhaustion in the culture medium. The intracellular NADP+ and protein concentrations increased in parallel during the exponential phase but were poorly correlated. Even after carbon exhaustion, the intracellular NADP+concentration stayed high, as did protein levels. The results demonstrated that the intracellular isocitrate concentration, but not the intracellular NADP+ concentration, was affected by the carbon availability in the culture. They also suggest that, because of its variability, isocitrate, but not NADP+, plays the larger role in the control of the respiratory CO2production rate (R CO2 )....
Microbial Physiology, 2005
During aerobic growth of Escherichia coli on acetate as sole source of carbon and energy, the organism requires the operation of the glyoxylate bypass enzymes, namely isocitrate lyase (ICL) and the anaplerotic enzyme malate synthase (MS). Under these conditions, the glyoxylate bypass enzyme ICL is in direct competition with the Krebs cycle enzyme isocitrate dehydrogenase (ICDH) for their common substrate and although ICDH has a much higher affinity for isocitrate, flux of carbon through ICL is assured by virtue of high intracellular level of isocitrate and the reversible phosphorylation/inactivation of a large fraction of ICDH. Reversible inactivation is due to reversible phosphorylation catalysed by ICDH kinase/phosphatase, which harbours both catalytic activities on the same polypeptide. The catalytic activities of ICDH kinase/phosphatase constitute a moiety conserved cycle, require ATP and exhibit ‘zero-order ultrasensitivity’. The structural gene encoding ICDH kinase/phosphatase...
Journal of Biomolecular Structure and Dynamics, 2009
Isocitrate Dehydrogenase (ICD) catalyzes the oxidative decarboxylation reaction of 2R,3Sisocitrate to yield 2-oxoglutarate in the Tricarboxylic Acid (TCA) cycle. Two isoforms of NADP-specific ICDs with the E.C number 1.1.1.42 have been annotated in the organism Mycobacterium tuberculosis, monomeric ICD2 and dimeric ICD1. BLAST search against the Protein Data Bank (PDB) database shows a marked similarity between dimeric Mycobacterium tuberculosis ICD1 sequence and that of Sus scrofa, a cytosolic eukaryotic ICD (65% identity). Escherischia coli ICD shows less sequence similarity than the eukaryotic structure. A Homology model has thus been built for M. tuberculosis ICD1 using Sus scrofa and human ICD as templates. Inactivation of ICD1 by phosphorylation similar to E. coli ICD is important to open up the shunt pathway in the TCA cycle, which has been indicated in the case of M. tuberculosis. We therefore attempted to identify a number of likely phosphorylation sites in M. tuberculosis using pattern prediction and checked with the homology models for the accessibility of the peptides containing Serine. It was found that the homologous Serine by alignment with E. coli on M. tuberculosis ICD1 is difficult to access by specific kinases. Hence other probable sites of phosphorylation were checked and three highly probable serine-containing peptides were identified. The effect of phosphorylation at each of these sites was determined by checking the degree of conformational changes, the differences caused by the effect of phosphorylation in the active-site and other apparent motion different from that of the control, i.e., unphosphorylated M. tuberculosis ICD1 model, using molecular dynamics simulations.
FEBS Letters, 1984
NADP+ can protect active isocitrate dehydrogenase against attack by several proteases. Inactive phosphorylated isocitrate dehydrogenase is much less susceptible to proteolysis than the active enzyme, and it is not protected by NADP+. The results suggest that binding of NADP+ to, or phosphorylation of, active isocitrate dehydrogenase induces similar conformational states. Fluorescence titration experiments show that NADPH can bind to active but not to inactive isocitrate dehydrogenase. It is suggested that the phosphorylation of isocitrate dehydrogenase may occur close to its coenzyme binding site.
Biochemistry, 2013
Mycobacterium tuberculosis (Mtb) is the leading cause of death due to a bacterial infection. The success of the Mtb pathogen has largely been attributed to the nonreplicating, persistence phase of the life cycle, for which the glyoxylate shunt is required. In Escherichia coli flux through the shunt is controlled by regulation of isocitrate dehydrogenase (ICDH). In Mtb, the mechanism of regulation is unknown, and currently there is no mechanistic or structural information on ICDH. We optimized expression and purification to a yield high enough to perform the first detailed kinetic and structural studies for Mtb ICDH-1. A large solvent kinetic isotope effect (D2O V = 3.0 ± 0.2, D2O [V/K isocitrate ] = 1.5 ± 0.3) and a smaller primary kinetic isotope effect (D V = 1.3 ± 0.1, D [V/K [2R-2 H]isocitrate ] = 1.5 ± 0.2) allowed us to perform the first multiple kinetic isotope effect studies on any ICDH and suggest a chemical mechanism. In this mechanism, protonation of the enolate to form product α-ketoglutarate is the rate-limiting step. We report the first structure of Mtb ICDH-1 to 2.18 Å by X-ray crystallography with NADPH and Mn 2+ bound. It is a homodimer in which each subunit has a Rossmann fold, and a common top domain of interlocking beta sheets. Mtb ICDH-1 is most structurally similar to the R132H mutant human ICDH found in glioblastomas. Similar to human R132H ICDH, Mtb ICDH-1 also catalyses the formation of αhydroxyglutarate. Our data suggest that regulation of Mtb ICDH-1 is novel.
Journal of Biological Chemistry, 2001
With the aim of gaining insight into the molecular and phylogenetic relationships of isocitrate dehydrogenase (IDH) from hyperthermophiles, we carried out a comparative study of putative IDHs identified in the genomes of the eubacterium Thermotoga maritima and the archaea Aeropyrum pernix and Pyrococcus furiosus. An optimum for activity at 90°C or above was found for each IDH. PfIDH and ApIDH were the most thermostable with a melting temperature of 103.7 and 109.9°C, respectively, compared with 98.3 and 98.5°C for TmIDH and AfIDH, respectively. Analytical ultracentrifugation revealed a tetrameric oligomeric state for TmIDH and a homodimeric state for ApIDH and PfIDH. TmIDH and ApIDH were NADP-dependent (K m(NADP) of 55.2 and 44.4 M, respectively) whereas PfIDH was NAD-dependent (K m(NAD) of 68.3 M). These data document that TmIDH represents a novel tetrameric NADP-dependent form of IDH and that PfIDH is a homodimeric NAD-dependent IDH not previously found among the archaea. The homodimeric NADP-IDH present in A. pernix is the most common form of IDH known so far. The evolutionary relationships of ApIDH, PfIDH, and TmIDH with all of the available amino acid sequences of di-and multimeric IDHs are described and discussed.