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PubMed1998

Fiber-type-related differences in the enzymes of a proposed substrate cycle.

A substrate cycle between citric acid cycle (CAC) intermediates isocitrate and 2-oxoglutarate, involving NAD+- and NADP+-linked isocitrate dehydrogenase (NAD-IDH and NADP-IDH, respectively) and mitochondrial transhydrogenase (H+-Thase), has recently been proposed. This cycle has been hypothesized to enhance mitochondrial respiratory control by increasing the sensitivity of NAD-IDH to its modulators and allowing for enhanced increases in flux through this step of the CAC during periods of increased ATP demand. The activities of the enzymes comprising the substrate cycle: NAD-IDH, forward and reverse NADP-IDH, and forward and reverse H+-Thase, along with the activity of a marker of mitochondrial content, citrate synthase (CS) were measured in mitochondria isolated from rabbit Type I and Type IIb muscles and in whole muscle homogenates, representing the various fiber types, from rats. In isolated rabbit muscle mitochondria, NAD-IDH had significantly higher (1.6 x ) activity in white muscle while forward NADP-IDH, forward and reverse H+-Thase, and CS all had significantly higher (1.2-1.6 x ) activities in red muscle. There was no difference in reverse NADP-IDH between fiber types. Similarly, in rat whole muscle enzyme activities normalized to CS, NAD-IDH had significantly higher activity in fast-twitch glycolytic (FG) fibers, while forward NADP-IDH and forward H+-Thase had significantly higher activities in slow-twitch oxidative (SO) fibers. These results suggest that differences in the activities of the substrate cycle enzymes between skeletal muscle fiber types could contribute to differences in respiratory control due to differential cycling rates and/or loci of control.

Animals↗

PubMed2005

Analysis of interactions with mitochondrial mRNA using mutant forms of yeast NAD(+)-specific isocitrate dehydrogenase.

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an allosterically regulated tricarboxylic acid cycle enzyme that has been shown to bind specifically and with high affinity to 5'-untranslated regions of yeast mitochondrial mRNAs. The absence of IDH has been shown to result in reduced expression of mitochondrial translation products, leading to the suggestion that this macromolecular interaction may contribute to regulating rates of translation. The interaction with mitochondrial mRNAs also produces a dramatic inhibition of IDH catalytic activity that is specifically alleviated by AMP, the primary allosteric activator of IDH. Using mutant forms of IDH with defined catalytic or regulatory kinetic defects, we found that residue changes altering ligand binding in the catalytic site reduce the inhibitory effect of a transcript from the mitochondrial COX2 mRNA. In contrast, residue changes altering binding of allosteric regulators do not prevent inhibition by the COX2 RNA transcript but do prevent alleviation of inhibition by AMP. Results obtained using surface plasmon resonance methods suggest that the mRNA transcript may bind at the active site of IDH. Also, the presence of AMP has little effect on overall affinity but renders the binding of mRNA ineffective in catalytic inhibition of IDH. Finally, by expressing mutant forms of IDH in vivo, we determined that detrimental effects on levels of mitochondrial translation products correlate with a substantial reduction in catalytic activity. However, concomitant loss of IDH and of citrate synthase eliminates these effects, suggesting that any role of IDH in mitochondrial translation is indirect.

Catalysis↗

PubMed2000

Bovine NAD+-dependent isocitrate dehydrogenase: alternative splicing and tissue-dependent expression of subunit 1.

NAD+-dependent isocitrate dehydrogenase (IDH), a key regulatory enzyme in the Krebs cycle, is a multi-tetrameric enzyme. At least three of the subunits in the core tetramer of mammals are unique gene products. Subunits 1/beta and 2/gamma are considered to be regulatory, while subunits 3,4/alpha, comprising half the tetramer, are catalytic. The full sequence was obtained for the major subunit 1 cDNA in bovine heart, IDH 1-A. A second cDNA, rare in heart, was also identified (IDH 1-B). Differences in the two were confined to the 3'-region, suggesting alternative splicing. Screening of brain, kidney, and liver RNA showed the presence of IDH 1-A and 1-B and a third major species, IDH 1-C. Amplification of bovine genomic DNA by PCR across the regions of difference produced a single product. Comparison of the genomic and mRNA sequences showed that IDH 1-A resulted from splicing of exon W to exon Y, eliminating intron w, exon X, and intron x. IDH 1-B was formed by splice junctions between exon W, exon X, and exon Y. IDH 1-C resulted from splicing of exon W to exon X and subsequent retention of intron x. The 2 proteins predicted from these 3 mRNAs are identical over their first 357 residues. Protein IDH 1-A, resulting from a termination codon within exon Y, contains an additional 26 residues. Proteins IDH 1-B and 1-C derive from a common termination codon within exon X and contain an additional 28 residues. The two C-terminal regions differ notably in the number and nature of charged residues, resulting in proteins with a charge difference of 3.2 at pH 7.0. Subunit 1 sequences previously reported from other species grouped with one or the other of the bovine proteins. No evidence was found for alternative splicing in subunit 3,4/alpha. The results of the present study, together with recent work on the 2/gamma subunit [Brenner,V., Nyakatura, G., Rosenthal, A., and Platzer, M. (1998) Genomics 44, 8], indicate that the regulatory subunits of the enzyme, but not the catalytic, possess alternatively spliced forms varying in C-terminal properties with tissue-specific expression. The finding is suggestive of a mechanism for modulation of allosteric regulation tailored to the needs of different tissues.

Alternative Splicing↗

PubMed2004

Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity.

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, and regulation of the enzymatic activity of IDHs is crucial for their biological functions. Bacterial IDHs are reversibly regulated by phosphorylation of a strictly conserved serine residue at the active site. Eukaryotic NADP-dependent IDHs (NADP-IDHs) have been shown to have diverse important biological functions; however, their regulatory mechanism remains unclear. Structural studies of human cytosolic NADP-IDH (HcIDH) in complex with NADP and in complex with NADP, isocitrate, and Ca2+ reveal three biologically relevant conformational states of the enzyme that differ substantially in the structure of the active site and in the overall structure. A structural segment at the active site that forms a conserved alpha-helix in all known NADP-IDH structures assumes a loop conformation in the open, inactive form of HcIDH; a partially unraveled alpha-helix in the semi-open, intermediate form; and an alpha-helix in the closed, active form. The side chain of Asp279 of this segment occupies the isocitrate-binding site and forms hydrogen bonds with Ser94 (the equivalent of the phosphorylation site in bacterial IDHs) in the inactive form and chelates the metal ion in the active form. The structural data led us to propose a novel self-regulatory mechanism for HcIDH that mimics the phosphorylation mechanism used by the bacterial homologs, consistent with biochemical and biological data. This mechanism might be applicable to other eukaryotic NADP-IDHs. The results also provide insights into the recognition and specificity of substrate and cofactor by eukaryotic NADP-IDHs.

Amino Acid Sequence↗

PubMed1976

Mitochondrial isocitrate dehydrogenase and isocitrate oxidation of rat ventral prostate.

Mitochondrial preparations isolated from rat ventral prostate were capable of oxidizing isocitrate by way of NADP isocitrate dehydrogenase (NADP-IDH) and NAD-IDH. NAD-IDH activity required ADP for activation. The pH responses for NAD-IDH and NADP-IDH were quite different. The results indicated that two different enzymes were involved in the NAD- and NADP-IDH activities. Indirect evidence indicated that NADPH-NAD transhydrogenase activity might also be involved in the mitochondrial pathway for isocitrate oxidation. NADP-IDH activity was significantly greater than NAD-IDH activity. The oxidation of isocitrate through IDH activity was coupled to the cytochrome system by NADPH- and NADH-cytochrome c reductase activities. Citrate, via isocitrate, oxidation proceeded at a much slower rate suggesting that aconitase activity could be limiting in the oxidation of citrate. In comparison to other tissues, the prostate oxidative enzyme activities are considerably lower. The results suggest that the accumulation of high prostate citrate levels is not due to a limitation imposed by a lack of IDH activity in prostate mitochondria.

Animals↗

PubMed1988

The isocitrate dehydrogenase phosphorylation cycle. Identification of the primary rate-limiting step.

In Escherichia coli, the branch point between the Krebs cycle and the glyoxylate bypass is regulated by the phosphorylation of isocitrate dehydrogenase (IDH). Phosphorylation inactivates IDH, forcing isocitrate through the bypass. This bypass is essential for growth on acetate but does not serve a useful function when alternative carbon sources, such as glucose or pyruvate, are also present. When pyruvate or glucose is added to a culture growing on acetate, the cells responded by dephosphorylating IDH and thus inhibiting the flow of isocitrate through the glyoxylate bypass. In an effort to identify the primary rate-limiting step in the response of IDH phosphorylation to alternative carbon sources, we have examined the response rates of congenic strains of E. coli which express different levels of IDH kinase/phosphatase, the bifunctional protein which catalyzes this phosphorylation cycle. The rate of the pyruvate-induced dephosphorylation of IDH was proportional to the level of IDH kinase/phosphatase, indicating that IDH kinase/phosphatase was primarily rate-limiting for dephosphorylation. However, the identity of the primary rate-limiting step appears to depend on the stimulus, since the rate of dephosphorylation of IDH in response to glucose was independent of the level of IDH kinase/phosphatase.

Escherichia coli↗

PubMed1985

[Isozymes of NADP-dependent isocitrate dehydrogenase in normal and tumor tissues of various mouse strains and their hybrids].

Normal tissues of DBA, CBA, CC57W, C3H, Balb/c, SHR mice and F1 hybrids CC57W/DBA appeared to differ in the ratios of mitochondrial and supernatant NADP-dependent isocitrate dehydrogenase (IDH). Tested inbred mice strains CC57W, C3H, SHR, Balb/c contain allelic form Idh-1a of supernatant IDH gene Idh-1, whereas allelic form Idh-1b is characteristic of mice strains DBA and CBA. In tumors IDH isozymes have the same mobility as do isozymes of homologous normal tissues; but their activity is lower. A high variability of each isozyme activity in the isozyme spectrum is revealed in various tissues of F1 hybrids CC57W/DBA. Allelic forms of gene Idh-1 were used as markers of normal and tumor cells for the experimental model: transplantation of sarcoma 37 (Idh-1a/Idh-1a) to subcutaneous tissue of the mouse strain DBA (Idh-1b/Idh-1b). It enables us to reveal isozymes of stromal cell in tumor IDH isozyme spectrum. The results indicate that the relation of normal and tumor isozymes vary in different tumors.

Alleles↗

PubMed1996

Molecular cloning of the cDNA of mouse mitochondrial NADP-dependent isocitrate dehydrogenase and the expression of the gene during lymphocyte activation.

The current report documents the molecular cloning of the mouse mitochondrial NADP-dependent isocitrate dehydrogenase (mNADP-IDH) cDNA. The cDNA was 1,863 bp in length and contained one open reading frame encoding a 523-residue polypeptide with a predicted molecular weight of 58 kDa. The cDNA and the deduced amino acid (AA) sequence of the mouse mNADP-IDH had a high degree of homology with those of porcine, bovine, alfalfa, and yeast. The recombinant mNADP-IDH expressed in Escherichia coli had active enzymatic function, as well as an expected molecular weight. The heart had the highest constitutive expression of the steady-state mNADP-IDH mRNA, followed by the kidney, while the expression of the gene in other tissues was low. The enzymatic activity of different tissues was in agreement with their mNADP-IDH mRNA levels. The resting lymphocytes had low constitutive expression of the gene, but the steady-state mRNA could be induced 48 h after mitogen stimulation. At the protein level, the resting lymphocytes had low enzymatic activity of mNADP-IDH, but the activity was augmented fivefold after mitogen stimulation. The cytosolic NADP-IDH, on the contrary, remained low or undetectable before and after the mitogen stimulation. Based on our current findings as well as the known roles of the mNADP-IDH in anabolism and in the isocitrate shuttle, it is conceivable that the mNADP-IDH is necessary for optimizing proliferation in lymphocytes.

Amino Acid Sequence↗

PubMed1977

Somatic cell genetic assignment of the human gene for mitochondrial NADP-linked isocitrate dehydrogenase to the long arm of chromosome 15.

A double-immunodiffusion method has been developed to detect human mitochondrial NADP-linked isocitrate dehydrogenase (EC 1.1.1.42; designated as IDH-2) using rabbit antiserum against the relevant enzyme. The method allows one to distinguish human IDH-2 from its mouse counterpart in extracts from human-mouse somatic cell hybrids. A correlation was found between the expression of human IDH-2 and the presence of human chromosome 15 in a "panel" of eight independent hybrid clones. Analysis of human marker enzymes for 37 different clones revealed a syntenic relationship between IDH-2 and mannose phosphate isomerase (EC 5.3.1.8; MPI), which has been assigned to chromosome 15 (1). These results permit the assignment of the structural gene for human IDH-2 to human chromosome 15. IDH-2 and human cytoplasmic IDH (IDH-1) were found to be asyntenic. Evidence from hybrid clones carrying a human X/15 translocation chromosome indicates that the human IDH-2 gene can be localized to the q11-qter region of chromosome 15.

Animals↗

PubMed2026

Methylome Profiling of Cartilage Tumors: A Promising New Diagnostic Tool?

DNA methylation and copy number variation (CNV) profiling has emerged as a promising tool for the classification of bone and soft tissue tumors. We evaluated its utility in cartilage tumors, where distinguishing low-grade from high-grade conventional central chondrosarcomas (CSs) and atypical cartilaginous tumors (ACTs) from enchondromas (ECs) is a frequent diagnostic challenge, particularly on biopsy material. We analyzed 214 chondrogenic tumors, including ECs, ACTs, conventional CSs, dedifferentiated chondrosarcomas (DDCSs), and clear cell CSs, and determined their IDH1/2 mutation status. Unsupervised dimensionality reduction of genome-wide DNA methylation patterns revealed 4 clusters among IDH-mutant (MUT) tumors (IDH-MUT-1: mostly ECs and ACTs and some high-grade CSs; IDH-MUT-2: predominantly high-grade CSs; IDH-MUT-3: largely DDCSs; and IDH-MUT-SB: distinct skull base group with a markedly different methylation pattern) and 2 clusters among IDH-wild-type (WT) tumors (IDH-WT-1 and IDH-WT-2: both primarily high-grade CSs, with IDH-WT-2 showing higher tumor grade and more extensive CNVs). Clear cell CSs formed a separate cluster. The amount of CNVs, including loss of CDKN2A, increased with tumor grade, reflecting increased genomic instability during chondrosarcoma progression. Supervised classifiers trained separately, both on methylation and CNV data, and distinguished low-grade and high-grade cartilaginous tumors with area under the curve values of 0.87 to 0.97 and 85% to 90% accuracy. Furthermore, we tested whether DDCSs can be distinguished from metastatic carcinomas and other high-grade sarcomas of the bone. Across 246 reference samples, a supervised classifier achieved 97.2% accuracy (area under the curve, 99.8%) and correctly identified 30 of 32 DDCSs (93.8%). These results indicate that DNA methylation and CNV data analysis provide a valuable tool for distinguishing most low- and high-grade CSs, with additional utility also in differentiating DDCS from morphologic mimics.

cartilaginous tumors↗

PubMed1998

Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants.

NAD-dependent isocitrate dehydrogenase (IDH) is a key enzyme controlling the activity of the citric acid cycle. Despite more than 30 years of work, the plant enzyme remains poorly characterized. In this paper, a molecular characterization of the plant IDH is presented. Starting from probes defined according to sequence comparisons, three full-length cDNAs named Ntidha, Ntidhb and Ntidhc encoding different IDH subunits have been isolated from a Nicotiana tabacum cell suspension library. Sequence comparisons of the tobacco IDH subunits with the E. coli NADP-dependent enzyme, and the yeast IDH1 and IDH2 subunits suggested that only IDHa had the capacity to be catalytic as IDHb and IDHc were lacking certain residues implied in catalysis. The ability of antibodies raised against the recombinant IDHa protein to preferentially cross-react with IDH2 indicated that IDHa was more closely related to IDH2 than to IDH1. Complementation of yeast single IDH mutants showed that IDHb and IDHc could replace the function of the yeast regulatory IDH1 subunit. Although IDHa was unable to complement the IDH2 mutant, its catalytic function was revealed by the ability of two heteromeric enzymes, composed of either IDHa with IDHb or IDHa with IDHc, to replace IDH function in a yeast double mutant lacking both subunits. Expression studies at the protein and mRNA levels show that each subunit is present in both root and leaf tissues and that the three IDH genes respond in the same way to nitrate addition. Taken together, such observations suggest that the physiologically active enzyme is composed of the three different subunits. These results show for the first time that the plant IDH is heteromeric and that IDH subunit composition appears to be conserved between plant and animal kingdoms.

Amino Acid Sequence↗

PubMed2002

Bacillus subtilis isocitrate dehydrogenase. A substrate analogue for Escherichia coli isocitrate dehydrogenase kinase/phosphatase.

In Escherichia coli, the homodimeric Krebs cycle enzyme isocitrate dehydrogenase (EcIDH) is regulated by reversible phosphorylation of a sequestered active site serine. The phosphorylation cycle is catalyzed by a bifunctional protein, IDH kinase/phosphatase (IDH-K/P). To better understand the nature of the interaction between EcIDH and IDH-K/P, we have examined the ability of an IDH homologue from Bacillus subtilis (BsIDH) to serve as a substrate for the kinase and phosphatase activities. BsIDH exhibits extensive sequence and structural similarities with EcIDH, particularly around the phosphorylated serine. Our previous crystallographic analysis revealed that the active site architecture of these two proteins is almost completely conserved. We now expand the comparison to include a number of biochemical properties. Both IDHs display nearly equivalent steady-state kinetic parameters for the dehydrogenase reaction. Both proteins are also phosphorylated by IDH-K/P in the same ratio (1 mole of phosphate per mole of monomer), and this stoichiometric phosphorylation correlates with an equivalent inhibition of IDH activity. Furthermore, tandem electrospray mass spectrometry demonstrates that BsIDH, like EcIDH, is phosphorylated on the corresponding active site serine residue (Ser-104). Despite the high degree of sequence, functional, and structural congruence between these two proteins, BsIDH is surprisingly a much poorer substrate of IDH-K/P than is EcIDH, with Michaelis constants for the kinase and phosphatase activities elevated by 60- and 3,450-fold, respectively. These drastically disparate values might result from restricted access to the active site cavity and/or from the lack of a potential docking site for IDH-K/P.

Bacillus subtilis↗

PubMed2005

Infradiaphragmatic versus supradiaphragmatic Hodgkin lymphoma: a retrospective review of 1,114 patients.

Infradiaphragmatic Hodgkin lymphoma (IDH) accounts for 4-13% of cases of stage I-II Hodgkin lymphoma (HD). It has been associated with distinct pre-treatment characteristics and outcomes when compared with supradiaphragmatic HD (SDH). The comparison of IDH vs SDH can only be made in early and intermediate stages (I-II), such a comparison is not possible for advanced stages (III-IV). This study retrospectively compared two groups of 1013 patients with stage I-II SDH and 101 patients with IDH (10%). These two sub-groups of patients were treated in 1988-1993 in 2 prospective randomized clinical trials in Germany for early and intermediate stages of Hodgkin lymphoma. IDH-patients were older (median 39 vs 31 years; p < 0.001), predominantly male (73% vs 52%; p < 0.001) and more often had involvement of 3 lymph node areas (LNA) (80% vs 55%; p < 0.001). Histology in IDH was more likely to be mixed cellularity (46.5% vs 23.6%, p < 0.001) or lymphocyte predominant (20 vs 10%, p = 0.003) and less likely nodular sclerosis (25% vs 63%, p < 0.001). In early-stage unfavorable disease, IDH was associated with a higher treatment failure rate (unadjusted hazard ratio 2, 95% CI, 1.3-3.4; p = 0.003). After controlling for age, sex, stage, histology, B-symptoms and involvement of 3 LNA, the adjusted hazard ratio was 1.25 (95% CI, 0.65-2.4; p = 0.51) so that IDH was no longer associated with a statistically significant treatment failure rate. Poorer outcomes with IDH as compared to SDH are attributable to its association with known adverse prognostic risk factors, but IDH, in itself, is not an independent adverse prognostic factor for treatment failure or survival.

Adult↗

PubMed2002

Isolated diastolic hypertension, pulse pressure, and mean arterial pressure as predictors of mortality during a follow-up of up to 32 years.

OBJECTIVE: To compare mortality associated with various blood pressure components in middle-aged men during up to 32 years of follow-up. DESIGN: A prospective cohort study. SETTING: Helsinki, Finland. PARTICIPANTS: We studied 3267 initially healthy men, aged 30-45 years, who participated in health check-ups from 1964 onwards. MAIN OUTCOME MEASURES: Cox regression was used to relate baseline blood pressure components to all-cause (n = 701) and cardiovascular disease (CVD) mortality (n = 325). RESULTS: Systolic (SBP) and diastolic (DBP) blood pressures, pulse pressure and mean arterial pressure singly predicted CVD mortality. With SBP 160 mmHg and DBP 90 mmHg as cut-off values, four blood pressure subgroups were identified: normotension (n = 1919), isolated systolic hypertension (ISH, n = 17), isolated diastolic hypertension (IDH, n = 1013), and combined systolic and diastolic hypertension (SDH, n = 318). IDH was subdivided into IDH-1 with SBP 140-159 mmHg (n = 667) and IDH-2 with SBP less than 140 mmHg (n = 346). With normotension as reference, only SDH and IDH-1 predicted CVD mortality [relative risk (RR) 2.71, 95% confidence interval (CI) 2.00 to 3.66, and RR 1.39, 95% CI 1.04 to 1.87, respectively]. Risk with IDH-2 (RR 1.14, 95% CI 0.77 to 1.69) was not statistically significant. SDH and IDH-1, but not IDH-2, were also associated with increased all-cause mortality risk. Use of antihypertensive medication did not explain the results. CONCLUSION: These results demonstrate the often neglected role of SBP in predicting long-term CVD risk in middle-aged men. When SBP is less than 140 mmHg, IDH is not associated with significantly increased risk of mortality. Administrative guidelines, which affect population health, should also take due note of SBP.

Adult↗

PubMed2004

A randomized, controlled trial of albumin versus saline for the treatment of intradialytic hypotension.

Intradialytic hypotension (IDH) is the most common complication of hemodialysis. Symptomatic IDH requires the administration of fluid and often results in the early termination of dialysis, both of which may prevent adequate fluid removal. The optimal fluid for the treatment of IDH remains unknown. A randomized, double-blind, crossover trial was performed in 72 chronic hemodialysis patients to determine whether 5% albumin was more effective than normal saline for the treatment of IDH. The primary outcome measure was the percentage of target ultrafiltration achieved, which was defined as the actual ultrafiltration volume divided by target ultrafiltration volume. Secondary outcome measures included postdialysis BP, time to restore BP, recurrent IDH, and treatment failure (inability to restore BP with 750 ml of study fluid). The percentage of target ultrafiltration achieved was 0.84 +/- 0.17 for 5% albumin compared with 0.80 +/- 0.16 for saline (P = 0.14). The postdialysis systolic BP (121 +/- 19 mmHg versus 117 +/- 19 mmHg; P = 0.32), postdialysis diastolic BP (63 +/- 9 mmHg versus 61 +/- 9 mmHg; P = 0.33), volume of study fluid used to treat IDH (403 +/- 170 ml versus 428 +/- 191 ml; P = 0.34), time required to restore the BP (7.9 +/- 6.6 min versus 9.9 +/- 7.5 min; P = 0.09), total nursing time required to manage the hypotensive episode (15.1 +/- 7.2 min versus 15.9 +/- 7.3 min; P = 0.47), number of treatment failures (22% versus 24%; P = 1.0), and the frequency of recurrent IDH (36% versus 36%) were not significantly different when 5% albumin was used compared with saline. It is concluded that 5% albumin is no more effective than normal saline for the treatment of IDH in chronic hemodialysis patients. Normal saline should be used as the initial fluid for the treatment of IDH.

Aged↗

PubMed1998

Purification and characterization of NADP+-linked isocitrate dehydrogenase from scots pine . Evidence for different physiological roles of the enzyme in primary development

NADP+-isocitrate dehydrogenase (NADP+-IDH; EC 1.1.1.42) is involved in the supply of 2-oxoglutarate for ammonia assimilation and glutamate synthesis in higher plants through the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Only one NADP+-IDH form of cytosolic localization was detected in green cotyledons of pine (Pinus spp.) seedlings. The pine enzyme was purified and exhibited molecular and kinetic properties similar to those described for NADP+-IDH from angiosperm, with a higher catalytic efficiency (10(5) M-1 s-1) than the deduced efficiencies for GS and GOGAT in higher plants. A polyclonal antiserum was raised against pine NADP+-IDH and used to assess protein expression in the seedlings. Steady-state levels of NADP+-IDH were coordinated with GS during seed germination and were associated with GS/GOGAT enzymes during chloroplast biogenesis, suggesting that NADP+-IDH is involved in the provision of carbon skeletons for the synthesis of nitrogen-containing molecules. However, a noncoordinated pattern of NADP+-IDH and GS/GOGAT was observed in advanced stages of cotyledon development and in the hypocotyl. A detailed analysis in hypocotyl sections revealed that NADP+-IDH abundance was inversely correlated with the presence of GS, GOGAT, and ribulose-1,5-bisphosphate carboxylase/oxygenase but was associated with the differentiation of the organ. These results cannot be explained by the accepted role of the enzyme in nitrogen assimilation and strongly suggest that NADP+-IDH may have other, as-yet-unknown, biological functions.

Journal Article↗

PubMed2002

Crystallization and preliminary X-ray structure analysis of isocitrate dehydrogenase from two hyperthermophiles, Aeropyrum pernix and Thermotoga maritima.

Isocitrate dehydrogenase (IDH) catalyses the dehydrogenation and decarboxylation of isocitrate to alpha-ketoglutarate and CO(2) with NAD or NADP as cofactor. IDH from Aeropyrum pernix is the most thermostable IDH identified. Crystals of A. pernix IDH diffracted to 2.6 A with synchrotron radiation and belong to space group P4(3)2(1)2. IDH from Thermotoga maritima is the only IDH that has been characterized as homotetrameric and might be an evolutionary link between two different IDH subfamilies. T. maritima IDH crystals diffracted to 2.8 A with Cu Kalpha radiation and belong to space group P2(1)2(1)2(1). The structures will be helpful in the study of the factors responsible for thermostability and the evolutionary relationships of IDHs.

Crystallization↗

PubMed1993

Genes encoding two isocitrate dehydrogenase isozymes of a psychrophilic bacterium, Vibrio sp. strain ABE-1.

The genes coding for two structurally different isocitrate dehydrogenase isozymes (IDH-I and IDH-II) of a psychrophilic bacterium, Vibrio sp. strain ABE-1, were cloned and sequenced. Open reading frames of the genes (icdI and icdII) are 1,248 and 2,229 bp in length, respectively. The amino acid sequences predicted from the open reading frames of icdI and icdII corresponded to the N-terminal amino acid sequences of the purified IDH-I and IDH-II, respectively. No homology was found between the deduced amino acid sequences of the isozymes; however, the IDH-I, a dimeric enzyme, had a high amino acid sequence identity (74.3%) to the Escherichia coli IDH. The deduced amino acid sequence of the IDH-II, a monomeric enzyme, was not related to any known sequence. However, the IDH-II had an amino acid sequence homologous to that of a cyanogen bromide-cleaved peptide containing a putative active-site methionyl residue of the monomeric IDH of Azotobacter vinelandii. The two genes (icdlI and icdII) were found to be tandemly located in the same orientation. Northern (RNA) blot analyses showed that the two genes are transcribed independently. Primer extension experiments located single transcriptional start sites 39 and 96 bp upstream of the start codons of icdI and icdII, respectively. The amount of icdI transcript but not icdII increased when Vibrio sp. strain ABE-1 cells were cultured in acetate minimal medium.

Amino Acid Sequence↗