Two isocitrate dehydrogenases from a psychrophilic bacterium, Colwellia psychrerythraea

Two structurally different monomeric and dimeric types of isocitrate dehydrogenase (IDH; EC 1.1.1.42) isozymes were confirmed to exist in a psychrophilic bacterium, Colwellia psychrerythraea, by Western blot analysis and the genes encoding them were cloned and sequenced. Open reading frames of the genes (icd-M and icd-D) encoding the monomeric and dimeric IDHs of this bacterium, IDH-M and IDH-D, were 2,232 and 1,251 bp in length and corresponded to polypeptides composed of 743 and 416 amino acids, respectively. The deduced amino acid sequences of the IDH-M and IDH-D showed high homology with those of monomeric and dimeric IDHs from other bacteria, respectively. Although the two genes were located in tandem, icd-M then icd-D, on the chromosomal DNA, a Northern blot analysis and primer extension experiment revealed that they are transcribed independent of each other. The expression of the monomeric and dimeric IDH isozyme genes in C. maris, a psychrophilic bacterium of the same genus as C. psychrerythraea, is known to be induced by low temperature and acetate, respectively, but no such induction in the expression of the C. psychrerythraea icd-M and icd-D genes was detected. IDH-M and IDH-D overexpressed in Escherichia coli were purified and characterized. In C. psychrerythraea, the IDH-M isozyme is cold-active whereas IDH-D is mesophilic, which is similar to C. maris that contains both cold-adapted and mesophilic isozymes of IDH. Experiments with chimeric enzymes between the cold-adapted monomeric IDHs of C. psychrerythraea and C. maris (IDH-M and ICD-II, respectively) suggested that the C-terminal region of the C. maris IDH-II is involved in its catalytic activity.     

A highly specific monomeric isocitrate dehydrogenase from Corynebacterium glutamicum

The monomeric isocitrate dehydrogenase (IDH) of Corynebacterium glutamicum is compared to the topologically distinct dimeric IDH of Escherichia coli. Both IDHs have evolved to efficiently catalyze identical reactions with similar pH optimum as well as striking specificity toward NADP and isocitrate. However, the monomeric IDH is 10-fold more active (calculated as kcat/Km.isocitrate/Km.NADP) and 7-fold more NADP-specific than the dimeric enzyme, favoring NADP over NAD by a factor of 50,000. Such an extraordinary coenzyme specificity is not rivaled by any other characterized dehydrogenases. In addition, the monomeric enzyme is 10-fold more specific for isocitrate. The spectacular substrate specificity may be predominantly attributed to the isocitrate-assisted stabilization of catalytic complex during hydride transfer. No significant overall sequence identity is found between the monomeric and dimeric enzymes. However, structure-based alignment leads to the identification of three regions in the monomeric enzyme that match closely the three motifs located in the central region of dimeric IDHs and the homologous isopropylmalate dehydrogenases. The role of Lys253 as catalytic residue has been demonstrated by site-directed mutagenesis. Our results suggest that monomeric and dimeric forms of IDHs are functionally and structurally homologous.     

Substrate-free structure of a monomeric NADP isocitrate dehydrogenase: an open conformation phylogenetic relationship of isocitrate dehydrogenase

Both monomeric and dimeric NADP+-dependent isocitrate dehydrogenase (IDH) belong to the metal-dependent beta-decarboxylating dehydrogenase family and catalyze the oxidative decarboxylation from 2R,3S-isocitrate to yield 2-oxoglutarate, CO2, and NADPH. It is important to solve the structures of IDHs from various species to correlate with its function and evolutionary significance. So far, only two crystal structures of substrate/cofactor-bound (isocitrate/NADP) NADP+-dependent monomeric IDH from Azotobacter vinelandii (AvIDH) have been solved. Herein, we report for the first time the substrate/cofactor-free structure of a monomeric NADP+-dependent IDH from Corynebacterium glutamicum (CgIDH) in the presence of Mg2+. The 1.75 A structure of CgIDH-Mg2+ showed a distinct open conformation in contrast to the closed conformation of AvIDH-isocitrate/NADP+ complexes. Fluorescence studies on CgIDH in the presence of isocitrate/or NADP+ suggest the presence of low energy barrier conformers. In CgIDH, the amino acid residues corresponding to the Escherichia coli IDH phosphorylation-loop are alpha-helical compared with the more flexible random-coil region in the E. coli protein where IDH activation is controlled by phosphorylation. This more structured region supports the idea that activation of CgIDH is not controlled by phosphorylation. Monomeric NADP+-specific IDHs have been identified from about 50 different bacterial species, such as proteobacteria, actinobacteria, and planctomycetes, whereas, dimeric NADP+-dependent IDHs are diversified in both prokaryotes and eukaryotes. We have constructed a phylogenetic tree based on amino acid sequences of all bacterial monomeric NADP+-dependent IDHs and also another one with specifically chosen species which either contains both monomeric and dimeric NADP+-dependent IDHs or have monomeric NADP+-dependent, as well as NAD+-dependent IDHs. This is done to examine evolutionary relationships.     

Structure of the monomeric isocitrate dehydrogenase: evidence of a protein monomerization by a domain duplication

NADP(+)-dependent isocitrate dehydrogenase is a member of the beta-decarboxylating dehydrogenase family and catalyzes the oxidative decarboxylation reaction from 2R,3S-isocitrate to yield 2-oxoglutarate and CO(2) in the Krebs cycle. Although most prokaryotic NADP(+)-dependent isocitrate dehydrogenases (IDHs) are homodimeric enzymes, the monomeric IDH with a molecular weight of 80-100 kDa has been found in a few species of bacteria. The 1.95 A crystal structure of the monomeric IDH revealed that it consists of two distinct domains, and its folding topology is related to the dimeric IDH. The structure of the large domain repeats a motif observed in the dimeric IDH. Such a fusional structure by domain duplication enables a single polypeptide chain to form a structure at the catalytic site that is homologous to the dimeric IDH, the catalytic site of which is located at the interface of two identical subunits.     

Crystal structure of the monomeric isocitrate dehydrogenase in the presence of NADP+: insight into the cofactor recognition,catalysis, and evolution

NADP+-dependent monomeric isocitrate dehydrogenase (IDH) from the nitrogen-fixing bacterium Azotobacter vinelandii (AvIDH) is one of members of the beta-decarboxylating dehydrogenase family and catalyzes the dehydration and decarboxylation of isocitrate to yield 2-oxoglutrate and CO2 in the Krebs cycle. We solved the crystal structure of the AvIDH in complex with cofactor NADP+ (AvIDH-NADP+ complex). The final refined model shows the closed form that has never been detected in any previously solved structures of beta-decarboxylating dehydrogenases. The structure also reveals all of the residues that interact with NADP+. The structure-based sequence alignment reveals that these residues were not conserved in any other dimeric NADP+-dependent IDHs. Therefore the NADP+ specificity of the monomeric and dimeric IDHs was independently acquired through the evolutional process. The AvIDH was known to show an exceptionally high turnover rate. The structure of the AvIDH-NADP+ complex indicates that one loop, which is not present in the Escherichia coli IDHs, reliably stabilizes the conformation of the nicotinamide mononucleotide of the bound NADP+ by forming a few hydrogen bonds, and such interactions are considered to be important for the monomeric enzyme to initiate the hydride transfer reaction immediately. Finally, the structure of the AvIDH is compared with that of other dimeric NADP-IDHs. Several structural features demonstrate that the monomeric IDHs are structurally more related to the eukaryotic dimeric IDHs than to the bacterial dimeric IDHs.     

Characterization of Chimeric Isocitrate Dehydrogenases of a Mesophilic Nitrogen-Fixing Bacterium, <Emphasis Type="Italic">Azotobacter vinelandii,</Emphasis> and a Psychrophilic Bacterium, <Emphasis Type="Italic">Colwellia maris</Emphasis>

Several properties of chimeric enzymes between a mesophilic isocitrate dehydrogenase (IDH) from a nitrogen-fixing bacterium, Azotobacter vinelandii, and a cold-adapted IDH isozyme (IDH-II) from a psychrophilic bacterium, Colwellia maris, were examined. Each of the genes encoding the IDHs was divided into four regions of almost equal lengths, and each region was ligated with different combinations to construct various chimeric genes. The resultant wild-type and chimeric genes were overexpressed in Escherichia coli. The wild-type and chimeric IDHs were classified into three groups based on optimum temperatures for activity of 20°, 30°, and 40°C. The IDHs with a lower optimum temperature were more thermolabile. The optimum temperature and thermostability of the chimeric enzymes decreased on increasing the proportion derived from the cold-adapted IDH-II of C. maris. Furthermore, the C-terminal region of the C. maris IDH-II was suggested to be responsible for its psychrophilic characteristics.     

Biochemical and phylogenetic characterization of a monomeric isocitrate dehydrogenase from a marine methanogenic archaeon Methanococcoides methylutens

Extremophiles - Monomeric isocitrate dehydrogenase (IDH) stands for a separated subgroup among IDH protein family. Up to now, all reported monomeric IDHs are from prokaryotes. Here, a monomeric IDH...     

Isozymes of isocitrate dehydrogenase from an obligately psychrophilic bacterium, Vibrio sp. strain ABE-1: purification, and modulation of activities by growth conditions

Each of the two isozymes, which are different in thermostability and quaternary structure, of isocitrate dehydrogenase (NADP+) [IDH: EC 1.1.1.42] was purified to an electrophoretically homogeneous state from an obligately psychrophilic marine bacterium, Vibrio sp. strain ABE-1. Hydrophobic chromatography was an efficient procedure to separate the two isozymes from each other. The isoelectric points of isozyme I (IDH-I; a dimer, Mr 88,100) and isozyme II (IDH-II; a monomer, Mr 80,500) were found to be pH 4.9 and 5.2, respectively. The two isozymes were similar in amino acid compositions, though there were slight differences in the contents of nonpolar and hydroxyl amino acids. However, their NH2-terminal amino acid sequences and immunochemical properties were clearly different from each other. The NH2-terminal amino acid sequence analysis also indicated that the subunits of IDH-I are chemically identical or highly homologous. Non-immuno-crossreactivity between the isozymes enabled us to measure the intracellular contents of the isozymes. IDH-I and -II were found to be differentially regulated in vivo by various growth conditions. IDH-I was induced by acetate, while IDH-II remained almost unchanged.     

The crystal structure of a hyperthermostable subfamily II isocitrate dehydrogenase from Thermotoga maritima

Isocitrate dehydrogenase (IDH) from the hyperthermophile Thermotoga maritima (TmIDH) catalyses NADP+- and metal-dependent oxidative decarboxylation of isocitrate to alpha-ketoglutarate. It belongs to the beta-decarboxylating dehydrogenase family and is the only hyperthermostable IDH identified within subfamily II. Furthermore, it is the only IDH that has been characterized as both dimeric and tetrameric in solution. We solved the crystal structure of the dimeric apo form of TmIDH at 2.2 A. The R-factor of the refined model was 18.5% (R(free) 22.4%). The conformation of the TmIDH structure was open and showed a domain rotation of 25-30 degrees compared with closed IDHs. The separate domains were found to be homologous to those of the mesophilic mammalian IDHs of subfamily II and were subjected to a comparative analysis in order to find differences that could explain the large difference in thermostability. Mutational studies revealed that stabilization of the N- and C-termini via long-range electrostatic interactions were important for the higher thermostability of TmIDH. Moreover, the number of intra- and intersubunit ion pairs was higher and the ionic networks were larger compared with the mesophilic IDHs. Other factors likely to confer higher stability in TmIDH were a less hydrophobic and more charged accessible surface, a more hydrophobic subunit interface, more hydrogen bonds per residue and a few loop deletions. The residues responsible for the binding of isocitrate and NADP+ were found to be highly conserved between TmIDH and the mammalian IDHs and it is likely that the reaction mechanism is the same.     

Isocitrate dehydrogenase isozymes from a psychrotrophic bacterium, Pseudomonas psychrophila

The genes encoding monomer- and dimer-type isocitrate dehydrogenase (IDH) isozymes from a psychrotrophic bacterium, Pseudomonas psychrophila, were cloned and sequenced. Open reading frames of the genes were 2,226 and 1,257 bp in length and corresponded to polypeptides composed of 741 and 418 amino acids, respectively. The deduced amino acid sequences showed high sequence identity with those of psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea, (about 70% identity) and the respective types of the putative IDH genes from other bacteria of genus Pseudomonas (more than 80% identity). The two genes were located in opposite direction from each other with a spacer of 463 bases in the order of dimeric and monomeric IDH genes on the chromosomal DNA, but analyses of northern blotting and 5′-terminal regions of the mRNAs revealed that they are transcribed independently. The expression of monomer- and dimer-type IDH genes in C. maris are known to be cold- and acetate-inducible, respectively, while only slight inductions by low temperature and/or acetate were observed in the expression of the P. psychrophila monomer- and dimer-type IDH genes. Both of these IDH isozymes overproduced in Escherichia coli showed mesophilic properties, in contrast with monomer- and dimer-type IDHs of C. maris as cold adapted and mesophilic enzymes, respectively. The substitution of Glu55 residue in the P. psychrophila monomeric IDH for Lys, which is the corresponding residue conserved between the cold-adapted monomeric IDHs from C. maris and C. psychrerythraea, by site-directed mutagenesis resulted in the decreased thermostability and the lowered optimum temperature of activity, suggesting that this residue is involved in the mesophilic properties of the P. psychrophila monomeric IDH.     

Characterization of NADP+-dependent isocitrate dehydrogenase isozymes from a psychrophilic bacterium,Colwellia psychrerythraea strain 34H

NADP⁺-dependent isocitrate dehydrogenase (IDH) isozymes of a psychrophilic bacterium, Colwellia psychrerythraea strain 34H, were characterized. The coexistence of monomeric and homodimeric IDHs in this bacterium was confirmed by Western blot analysis, the genes encoding two monomeric (IDH-IIa and IDH-IIb) and one dimeric (IDH-I) IDHs were cloned and overexpressed in Escherichia coli, and the three IDH proteins were purified. Both of the purified IDH-IIa and IDH-IIb were found to be cold-adapted enzymes while the purified IDH-I showed mesophilic properties. However, the specific activities of IDH-IIa and IDH-IIb were lower even at low temperatures than that of IDH-I. Therefore, IDH-I was suggested to be important for the growth of this bacterium. The results of colony formation of E. coli transformants carrying the respective IDH genes and IDH activities in their crude extracts indicated that the expression of the IDH-IIa gene is cold-inducible in the E. coli cells.     

Controlled specific expression and purification of 6×His-tagged proteins in Pseudomonas

The Coxiella burnetii icd gene encoding an immunogenic dimeric NADP(+)-dependent isocitrate dehydrogenase (IDH) was cloned by screening a C. burnetii genomic library with a human positive serum and sequenced. The predicted gene product consists of 427 amino acids (M(r) = 46,600) and showed high identity to the IDHs of Escherichia coli (74%), Salmonella enterica (73%) and IDH-I of Vibrio sp. (71%). The cloned gene complemented an icd-defective E. coli mutant producing a recombinant IDH that had the same biochemical properties as the enzyme from purified C. burnetii. Unlike the homologs from other bacteria, the cloned enzyme was expressed to the highest level in low pH conditions. This distinct property of the cloned IDH suggests that C. burnetii icd gene may have a role in the adaptation of the organism to the harsh acidic environment of the eucaryotic phagolysosomes.     

Characterization of isocitrate dehydrogenase from the green sulfur bacterium Chlorobium limicola. A carbon dioxide-fixing enzyme in the reductive tricarboxylic acid cycle.

Isocitrate dehydrogenase (IDH) catalyzes the reversible conversion between isocitrate and 2-oxoglutarate accompanied by decarboxylation/carboxylation and oxidoreduction of NAD(P)+ cofactor. While this enzyme has been well studied as a catabolic enzyme in the tricarboxylic acid (TCA) cycle, here we have characterized NADP-dependent IDH from Chlorobium limicola, a green sulfur bacterium that fixes CO2 through the reductive tricarboxylic acid (RTCA) cycle, focusing on the CO2-fixation ability of the enzyme. The gene encoding Cl-IDH consisted of 2226 bp, corresponding to a polypeptide of 742 amino acid residues. The primary structure and the size of the recombinant protein indicated that Cl-IDH was a monomeric enzyme of 80 kDa distinct from the dimeric NADP-dependent IDHs predominantly found in bacteria or eukaryotic mitochondria. Apparent Michaelis constants for isocitrate (45 +/- 13 microm) and NADP+ (27 +/- 10 microm) were much smaller than those for 2-oxoglutarate (1.1 +/- 0.5 mm) and CO2 (1.3 +/- 0.3 mm). No significant differences in kinetic properties were observed between Cl-IDH and the dimeric, NADP-dependent IDH from Saccharomyces cerevisiae (Sc-IDH) at the optimum pH of each enzyme. However, in contrast to the 20% activity of Sc-IDH toward carboxylation as compared with that toward decarboxylation at pH 7.0, the activities of Cl-IDH for both directions were almost equivalent at this pH, suggesting a more favorable property of Cl-IDH than Sc-IDH as a CO2-fixation enzyme under physiological pH. Furthermore, we found that among various intermediates, oxaloacetate was a competitive inhibitor (K(i) = 0.35 +/- 0.04 mm) for 2-oxoglutarate in the carboxylation reaction by Cl-IDH, a feature not found in Sc-IDH.     

NADP(+)-isocitrate dehydrogenase from the cyanobacterium Anabaena sp. strain PCC 7120: purification and characterization of the enzyme and cloning, sequencing, and disruption of the icd gene.

NADP(+)-isocitrate dehydrogenase (NADP(+)-IDH) from the dinitrogen-fixing filamentous cyanobacterium Anabaena sp. strain PCC 7120 was purified to homogeneity. The native enzyme is composed of two identical subunits (M(r), 57,000) and cross-reacts with antibodies obtained against the previously purified NADP(+)-IDH from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. Anabaena NADP(+)-IDH resembles in its physicochemical and kinetic parameters the typical dimeric IDHs from prokaryotes. The gene encoding Anabaena NADP(+)-IDH was cloned by complementation of an Escherichia coli icd mutant with an Anabaena genomic library. The complementing DNA was located on a 6-kb fragment. It encodes an NADP(+)-IDH that has the same mobility as that of Anabaena NADP(+)-IDH on nondenaturing polyacrylamide gels. The icd gene was subcloned and sequenced. Translation of the nucleotide sequence gave a polypeptide of 473 amino acids that showed high sequence similarity to the E. coli enzyme (59% identity) and with IDH1 and IDH2, the two subunits of the heteromultimeric NAD(+)-IDH from Saccharomyces cerevisiae (30 to 35% identity); however, a low level of similarity to NADP(+)-IDHs of eukaryotic origin was found (23% identity). Furthermore, Anabaena NADP(+)-IDH contains a 44-residue amino acid sequence in its central region that is absent in the other IDHs so far sequenced. Attempts to generate icd mutants by insertional mutagenesis were unsuccessful, suggesting an essential role of IDH in Anabaena sp. strain PCC 7120.     

Bacterial Adaptation to Low Temperature: Implications for Cold-Inducible Genes

Escherichia coli and later found to be a cold-shock response common to many bacterial species. CspA of 7.4 kD, a major cold-shock protein in E. coli, has been shown to share structural similarity with a class of eukaryotic Y box proteins which have RNA-binding domains. Transient synthesis of CspA upon cold shock is mediated by increased stabilization of the mRNA at low temperatures. The proposed role of some cold-shock proteins including CspA in the bacterial adaptation to low temperatures is to function as a RNA chaperone in the regulation of translation. Some enzymes of psychrotrophic or psychrophilic bacteria exhibit unique features of a cold-adapted enzyme, high catalytic activity at a low temperature and rapid inactivation at a moderate temperature. A monomeric isocitrate dehydrogenase isozyme (IDH-II) of a psychrophilic bacterium, Vibrio sp. strain ABE-1, is a typical cold-adapted enzyme. In addition, this enzyme is induced at low temperatures. Low temperature-dependent expression of icdll encoding IDH-II is controlled by two different cis-elements located at the untranslated upstream region of the gene, one is a silencer and the other is essential for the low temperature response. The physiological role of IDH-II is evaluated by transforming E. coli with icdll. The growth rate of the E. coli transformants at low temperatures is dependent on the level of expressed IDH-II activity. Received 11 January 1999/ Accepted in revised form 6 April 1999     

Cloning,sequencing, and expression of a gene encoding the monomeric isocitrate dehydrogenase of the nitrogen-fixing bacterium,Azotobacter vinelandii

Isocitrate dehydrogenase (IDH: EC 1.1.1.42) of Azotobacter vinelandii was purified to an electrophoretically homogeneous state, and a gene (icd) encoding this enzyme was cloned and sequenced. The N-terminal amino acid sequence of the purified enzyme was consistent with that deduced from the nucleotide sequence of the icd gene. The deduced amino acid sequence of this gene showed high identity (62-66%) to those of the other bacterial monomeric IDHs. Expression of the icd gene in Escherichia coli was examined by measuring the enzyme activity and mRNA level. Primer extension analyses revealed that two species of mRNAs with different lengths of 5'-untranslated regions (TS-1 and TS-2) were present, of which the 5'-terminals (TS-1 and TS-2 sites) were cytosines located at 244 bp and 101 bp upstream of translational initiation codon, respectively. Conserved promoter elements were present at -35 and -10 regions from the TS-1 site, whereas no such a common motif was found in the upstream region of the TS-2 site. Deletion of the promoter elements upstream of the TS-1 site resulted in complete loss of IDH activity in the E. coli transformant. When the promoter elements upstream of the TS-1 site were intact, the levels of TS-1 and TS-2 were varied greatly by altering exogenous nutrients for growth. The cells grown in a nutrient-rich medium produced large amounts of TS-1 and had a low level of IDH activity. In a nutrient-poor medium, the cells contained large amounts of TS-2 and high levels of IDH activity.     

Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme.

NADP(+)-dependent isocitrate dehydrogenase (ICD) is an important enzyme of the intermediary metabolism, as it controls the carbon flux within the citric acid cycle and supplies the cell with 2-oxoglutarate and NADPH for biosynthetic purposes. In the amino acid-producing organism Corynebacterium glutamicum, the specific activity of ICD was independent of the growth substrate and of the growth phase at approximately 1 U/mg, indicating that this enzyme is constitutively formed. The ICD gene, icd, was isolated, subcloned on a plasmid, and introduced into C. glutamicum. Compared with the wild type, the recombinant strains showed up to 10-fold-higher specific ICD activities. The nucleotide sequence of a 3,595-bp DNA fragment containing the icd gene was determined. The predicted gene product of icd consists of 739 amino acids (M(r) = 80.091) and showed 58.5% identity with the monomeric ICD isozyme II from Vibrio sp. strain ABE-1 but no similarity to any known ICD of the dimeric type. Inactivation of the chromosomal icd gene led to glutamate auxotrophy and to the absence of any detectable ICD activity, suggesting that only a single ICD is present in C. glutamicum. From an icd-overexpressing C. glutamicum strain, ICD was purified and biochemically characterized. The native ICD was found to be a monomer; to be specific for NADP+; to be weakly inhibited by oxaloacetate, 2-oxoglutarate, and citrate; and to be severely inhibited by oxaloacetate plus glyoxylate. The data indicate that ICD from C. glutamicum is structurally similar to ICDs from bacteria of the genera Vibrio, Rhodomicrobium, and Azotobacter but different from all other known procaryotic and eucaryotic ICDs.     

Isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix: X-ray structure analysis of a ternary enzyme-substrate complex and thermal stability

Isocitrate dehydrogenase from Aeropyrum pernix (ApIDH) is a homodimeric enzyme that belongs to the beta-decarboxylating dehydrogenase family and is the most thermostable IDH identified. It catalyzes the NADP+ and metal-dependent oxidative decarboxylation of isocitrate to alpha-ketoglutarate. We have solved the crystal structures of a native ApIDH at 2.2 A, a pseudo-native ApIDH at 2.1 A, and of ApIDH in complex with NADP+, Ca2+ and d-isocitrate at 2.3 A. The pseudo-native ApIDH is in complex with etheno-NADP+ which was located at the surface instead of in the active site revealing a novel adenine-nucleotide binding site in ApIDH. The native and the pseudo-native ApIDHs were found in an open conformation, whereas one of the subunits of the ternary complex was closed upon substrate binding. The closed subunit showed a domain rotation of 19 degrees compared to the open subunit. The binding of isocitrate in the closed subunit was identical with that of the binary complex of porcine mitochondrial IDH, whereas the binding of NADP+ was similar to that of the ternary complex of IDH from Escherichiacoli. The reaction mechanism is likely to be conserved in the different IDHs. A proton relay chain involving at least five solvent molecules, the 5'-phosphate group of the nicotinamide-ribose and a coupled lysine-tyrosine pair in the active site, is postulated as essential in both the initial and the final steps of the catalytic reaction of IDH. ApIDH was found to be highly homologous to the mesophilic IDHs and was subjected to a comparative analysis in order to find differences that could explain the large difference in thermostability. Mutational studies revealed that a disulfide bond at the N terminus and a seven-membered inter-domain ionic network at the surface are major determinants for the higher thermostability of ApIDH compared to EcIDH. Furthermore, the total number of ion pairs was dramatically higher in ApIDH compared to the mesophilic IDHs if a cutoff of 4.2 A was used. A calculated net charge of only +1 compared to -19 and -25 in EcIDH and BsIDH, respectively, suggested a high degree of electrostatic optimization, which is known to be an important determinant for increased thermostability.     

Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme

The gene encoding isocitrate dehydrogenase (IDH) of Methylococcus capsulatus (McIDH) was cloned and overexpressed in Escherichia coli. The purified enzyme was NAD⁺-dependent with a thermal optimum for activity at 55–60°C and an apparent midpoint melting temperature (T _m) of 70°C. Analytical ultracentrifugation (AUC) revealed a homotetrameric state, and McIDH thus represents the first homotetrameric NAD⁺-dependent IDH that has been characterized. Based on a structural alignment of McIDH and homotetrameric homoisocitrate dehydrogenase (HDH) from Thermus thermophilus (TtHDH), we identified the clasp-like domain of McIDH as a likely site for tetramerization. McIDH showed moreover, higher sequence identity (48%) to TtHDH than to previously characterized IDHs. Putative NAD⁺-IDHs with high sequence identity (48–57%) to McIDH were however identified in a variety of bacteria showing that NAD⁺-dependent IDHs are indeed widespread within the domain, Bacteria. Phylogenetic analysis including these new sequences revealed a close relationship with eukaryal allosterically regulated NAD⁺-IDH and the subfamily III of IDH was redefined to include bacterial NAD⁺- and NADP⁺-dependent IDHs. This apparent relationship suggests that the mitochondrial genes encoding NAD⁺-IDH are derived from the McIDH-like IDHs.     

Knockdown of both mitochondrial isocitrate dehydrogenase enzymes in pancreatic beta cells inhibits insulin secretion

Background

There are three isocitrate dehydrogenases (IDHs) in the pancreatic insulin cell; IDH1 (cytosolic) and IDH2 (mitochondrial) use NADP(H). IDH3 is mitochondrial, uses NAD(H) and was believed to be the IDH that supports the citric acid cycle.

Methods

With shRNAs targeting mRNAs for these enzymes we generated cell lines from INS-1 832/13 cells with severe (80%–90%) knockdown of the mitochondrial IDHs separately and together in the same cell line.

Results

With knockdown of both mitochondrial IDH's mRNA, enzyme activity and protein level, (but not with knockdown of only one mitochondrial IDH) glucose- and BCH (an allosteric activator of glutamate dehydrogenase)-plus-glutamine-stimulated insulin release were inhibited. Cellular levels of citrate, α-ketoglutarate, malate and ATP were altered in patterns consistent with blockage at the mitochondrial IDH reactions. We were able to generate only 50% knockdown of Idh1 mRNA in multiple cell lines (without inhibition of insulin release) possibly because greater knockdown of IDH1 was not compatible with cell line survival.

Conclusions

The mitochondrial IDHs are redundant for insulin secretion. When both enzymes are severely knocked down, their low activities (possibly assisted by transport of IDH products and other metabolic intermediates from the cytosol into mitochondria) are sufficient for cell growth, but inadequate for insulin secretion when the requirement for intermediates is certainly more rapid. The results also indicate that IDH2 can support the citric acid cycle.

General significance

As almost all mammalian cells possess substantial amounts of all three IDH enzymes, the biological principles suggested by these results are probably extrapolatable to many tissues.