Isocitrate dehydrogenase kinase/phosphatase: aceK alleles that express kinase but not phosphatase activity.

For Escherichia coli, growth on acetate requires the induction of the enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase. The branch point between the glyoxylate bypass and the Krebs cycle is controlled by phosphorylation of isocitrate dehydrogenase (IDH), inhibiting that enzyme's activity and thus forcing isocitrate through the bypass. This phosphorylation cycle is catalyzed by a bifunctional enzyme, IDH kinase/phosphatase, which is encoded by aceK. We have employed random mutagenesis to isolate novel alleles of aceK. These alleles were detected by the loss of ability to complement an aceK null mutation. The products of one class of these alleles retain IDH kinase activity but have suffered reductions in IDH phosphatase activity by factors of 200 to 400. Selective loss of the phosphatase activity also appears to have occurred in vivo, since cells expressing these alleles exhibit phenotypes which are reminiscent of strains lacking IDH; these strains are auxotrophic for glutamate. Assays of cell-free extracts confirmed that this phenotype resulted from nearly quantitative phosphorylation of IDH. The availability of these novel alleles of aceK allowed us to assess the significance of the precise control which is a characteristic of the IDH phosphorylation cycle in vivo. The fractional phosphorylation of IDH was varied by controlled expression of one of the mutant alleles, aceK3, in a wild-type strain. Reduction of IDH activity to 50% of the wild-type level did not adversely affect growth on acetate. However, further reductions inhibited growth, and growth arrest occurred when the IDH activity fell to 15% of the wild-type level. Thus, although wild-type cells maintain a precise effective IDH activity during growth on acetate, this precision is not critical.     

Isocitrate dehydrogenase kinase/phosphatase: identification of mutations which selectively inhibit phosphatase activity.

Mutations in aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase, which selectively inhibit phosphatase activity have been isolated. These mutations yield amino acid substitutions within a 113-residue region of this 578-residue protein. These mutations may define a regulatory domain of this protein.     

Locations of the regulatory sites for isocitrate dehydrogenase kinase/phosphatase

Isocitrate dehydrogenase (IDH)(1) of Escherichia coli is regulated by a bifunctional protein, IDH kinase/phosphatase. In this paper, we demonstrate that the effectors controlling these activities belong to two distinct classes that differ in mechanism and in the locations of their binding sites. NADPH and isocitrate are representative members of one of these effector classes. NADPH inhibits both IDH kinase and IDH phosphatase, whereas isocitrate inhibits only IDH kinase. Isocitrate can "activate" IDH phosphatase by reversing product inhibition by dephospho-IDH. Mutations in icd, which encodes IDH, had parallel effects on the binding of these ligands to the IDH active site and on their effects on IDH kinase and phosphatase, indicating that these ligands regulate IDH kinase/phosphatase through the IDH active site. Kinetic analyses suggested that isocitrate and NADPH prevent formation of the complex between IDH kinase/phosphatase and its protein substrate. AMP, 3-phosphoglycerate, and pyruvate represent a class of regulatory ligands that is distinct from that which includes isocitrate and NADPH. These ligands bind directly to IDH kinase/phosphatase, a conclusion which is supported by the observation that they inhibit the IDH-independent ATPase activity of this enzyme. These effector classes can also be distinguished by the observation that mutant derivatives of IDH kinase/phosphatase expressed from aceK3 and aceK4 exhibited dramatic changes in their responses to AMP, 3-phosphoglycerate, and pyruvate but not to NADPH and isocitrate.     

Isocitrate dehydrogenase of Tetrahymena pyriformis

Summary We have studied the isocitrate dehydrogenase ofTetrahymena pyriformis. This enzyme is able to utilize both NAD and NADP, but kinetic studies suggest that the enzymatic activity with NAD is not of physiological significance.Some of the factors that might regulate the NADP-dependent isocitrate dehydrogenase were also studied. This enzyme has an absolute requirement for divalent cations; Mg²⁺ and Mn²⁺ will serve as cofactors but the latter is more effective than the former.It is known that this enzyme is subject to a concerted inhibition by oxaloacetate and glyoxylate. Either glyoxylate or oxaloacetate alone also are capable of inhibiting the enzyme although higher concentrations are required. We have found concerted inhibition also for the NAD-dependent isocitrate dehydrogenase from rat liver and yeast. The activity of theTetrahymena pyriformis enzyme is inhibited by NADPH. This inhibition is competitive with NADP. The Ki and Km values are, respectively, 23µ m and 18µ m.     

Isocitrate dehydrogenase of Plasmodium falciparum.

Erythrocytic stages of the malaria parasite Plasmodium falciparum rely on glycolysis for their energy supply and it is unclear whether they obtain energy via mitochondrial respiration albeit enzymes of the tricarboxylic acid (TCA) cycle appear to be expressed in these parasite stages. Isocitrate dehydrogenase (ICDH) is either an integral part of the mitochondrial TCA cycle or is involved in providing NADPH for reductive reactions in the cell. The gene encoding P. falciparum ICDH was cloned and analysis of the deduced amino-acid sequence revealed that it possesses a putative mitochondrial targeting sequence. The protein is very similar to NADP+-dependent mitochondrial counterparts of higher eukaryotes but not Escherichia coli. Expression of full-length ICDH generated recombinant protein exclusively expressed in inclusion bodies but the removal of 27 N-terminal amino acids yielded appreciable amounts of soluble ICDH consistent with the prediction that these residues confer targeting of the native protein to the parasites' mitochondrion. Recombinant ICDH forms homodimers of 90 kDa and its activity is dependent on the bivalent metal ions Mg2+ or Mn2+ with apparent Km values of 13 micro m and 22 micro m, respectively. Plasmodium ICDH requires NADP+ as cofactor and no activity with NAD+ was detectable; the for NADP+ was found to be 90 micro m and that of d-isocitrate was determined to be 40 micro m. Incubation of P. falciparum under exogenous oxidative stress resulted in an up-regulation of ICDH mRNA and protein levels indicating that the enzyme is involved in mitochondrial redox control rather than energy metabolism of the parasites.     

Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase

The specificities of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase were probed using synthetic peptides corresponding to the sequence around phosphorylation sites 1 and 2 on pyruvate dehydrogenase [Tyr-His-Gly-His-Ser(P1)-Met-Ser-Asp-Pro-Gly-Val-Ser(P2)-Tyr-Arg]. The dephosphotetradecapeptide containing aspartic acid at position 8 was a better substrate for the kinase than was the tetradecapeptide containing asparagine at position 8. The apparent Km and V values for the two peptides were 0.43 and 6.1 mM and 2.7 and 2.4 nmol of 32P incorporated/min/mg, respectively. Methylation of the aspartic acid residue also increased the apparent Km of the tetradecapeptide about 14-fold. These results indicate that an acidic residue on the carboxyl-terminal side of phosphorylation site 1 is an important specificity determinant for the kinase. Phosphate was incorporated only into site 1 of the synthetic peptide by the kinase. The phosphatase exhibited an apparent Km of 0.28 mM and a V of 2.3 mumol of 32P released/min/mg for the phosphorylated tetradecapeptide containing aspartic acid. Methylation of the aspartic acid residue had no significant effect on dephosphorylation. The octapeptide and phosphooctapeptide produced by cleavage of the aspartyl-prolyl bond by formic acid were poorer substrates for the kinase and phosphatase than were the tetradecapeptide and phosphotetradecapeptide, respectively. Modification of the amino terminal by acetylation or lysine addition had only a slight effect on the kinase and phosphatase activities.     

Structural basis of the substrate specificity of bifunctional isocitrate dehydrogenase kinase/phosphatase

Isocitrate dehydrogenase kinase/phosphatase (AceK) regulates entry into the glyoxylate bypass by reversibly phosphorylating isocitrate dehydrogenase (ICDH). On the basis of the recently determined structure of the AceK-ICDH complex from Escherichia coli, we have classified the structures of homodimeric NADP(+)-ICDHs to rationalize and predict which organisms likely contain substrates for AceK. One example is Burkholderia pseudomallei (Bp). Here we report a crystal structure of Bp-ICDH that exhibits the necessary structural elements required for AceK recognition. Kinetic analyses provided further confirmation that Bp-ICDH is a substrate for AceK. We conclude that the highly stringent AceK binding sites on ICDH are maintained only in Gram-negative bacteria.     

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.     

Structural and mechanistic insights into the bifunctional enzyme isocitrate dehydrogenase kinase/phosphatase AceK

The switch between the Krebs cycle and the glyoxylate bypass is controlled by isocitrate dehydrogenase kinase/phosphatase (AceK). AceK, a bifunctional enzyme, phosphorylates and dephosphorylates isocitrate dehydrogenase (IDH) with its unique active site that harbours both the kinase and ATP/ADP-dependent phosphatase activities. AceK was the first example of prokaryotic phosphorylation identified, and the recent characterization of the structures of AceK and its complex with its protein substrate, IDH, now offers a new understanding of both previous and future endeavours. AceK is structurally similar to the eukaryotic protein kinase superfamily, sharing many of the familiar catalytic and regulatory motifs, demonstrating a close evolutionary relationship. Although the active site is shared by both the kinase and phosphatase functions, the catalytic residues needed for phosphatase function are readily seen when compared with the DXDX(T/V) family of phosphatases, despite the fact that the phosphatase function of AceK is strictly ATP/ADP-dependent. Structural analysis has also allowed a detailed look at regulation and its stringent requirements for interacting with IDH.     

Genetic polymorphism of Isocitrate dehydrogenase in Primates

Summary The genetic polymorphism of isocitrate dehydrogenase has been investigated in 400 Primates. Six different phenotypes of the soluble enzyme were demonstrated. The mitochondrial enzyme exhibited a uniform electrophoretic pattern.

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Zusammenfassung Die Isocitratdehydrogenasen der Primaten zeigen eine genetisch determinierte Variabilität. Für das cytoplasmatische Enzym konnten 6 verschiedene Phänotypen nachgewiesen werden. Das mitochondriale Enzym verhielt sich in der Elektrophorese einheitlich.

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Supported by the Deutsche Forschungsgemeinschaft.     

Nucleotide sequence of aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase.

In Escherichia coli, the phosphorylation and dephosphorylation of isocitrate dehydrogenase (IDH) are catalyzed by a bifunctional protein kinase/phosphatase. We have determined the nucleotide sequence of aceK, the gene encoding IDH kinase/phosphatase. This gene consists of a single open reading frame of 1,734 base pairs preceded by a Shine-Dalgarno ribosome-binding site. Examination of the deduced amino acid sequence of IDH kinase/phosphatase revealed sequences which are similar to the consensus sequence for ATP-binding sites. This protein did not, however, exhibit the extensive sequence homologies which are typical of other protein kinases. Multiple copies of the REP family of repetitive extragenic elements were found within the intergenic region between aceA (encoding isocitrate lyase) and aceK. These elements have the potential for combining to form an exceptionally stable stem-loop structure (delta G = -54 kcal/mol [ca. -226 kJ/mol]) in the mRNA. This structure, which masks the ribosome-binding site and start codon for aceK, may contribute to the downshift in expression observed between aceA and aceK. Another potential stem-loop structure (delta G = -29 kcal/mol [ca. 121 kJ/mol]), unrelated to the REP sequences, was found within aceK.     

Isocitrate dehydrogenase assays on intact bacterial cells

A qualitative assay which can be adapted to screen large numbers of Escherichia coli colonies for the presence of soluble enzymes is described. In a test of the system using a new, especially sensitive assay for isocitrate dehydrogenase activity, colonies producing the enzyme could be correctly identified at the 70% level after 2 h of incubation and at the 100% level after 8 h of incubation. The completed reactions are stable for several days at room temperature.     

Peptides derived from pyruvate dehydrogenase as substrates for pyruvate dehydrogenase kinase and phosphatase.

Evidence is presented that phosphopeptides produced by tryptic digestion of phosphorylated pyruvate dehydrogenase are effective substrates for pyruvate dehydrogenase phosphatase and that the dephosphopeptides can serve as substrates for pyruvate dehydrogenase kinase. These findings indicate that the phosphatase and the kinase do not require an intact tertiary structure in pyruvate dehydrogenase, but apparently recognize components of the local primary sequence around the phosphorylation sites.     

Photoaffinity labelling shows that Escherichia coli isocitrate dehydrogenase kinase/phosphatase contains a single ATP-binding site

Ultraviolet irradiation of E.coli isocitrate dehydrogenase kinase/phosphatase in the presence of 8-azidoATP resulted in parallel losses of its kinase and phosphatase activities, and in covalent attachment of the reagent to the protein at a single site. ATP and ADP protected the two activities to similar extents. The data suggest that the activation of the phosphatase by adenine nucleotides results from binding of the nucleotides to the active site of the kinase.     

Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase

In Escherichia coli, the reversible phosphorylation of isocitrate dehydrogenase (IDH) is catalyzed by a bifunctional protein: IDH kinase/phosphatase. Although both IDH kinase and IDH phosphatase require ATP, the amino acid sequence of IDH kinase/phosphatase contains a single sequence that matches the consensus for ATP binding sites. A mutation that converted the "invariant" lysine (residue 336) of this consensus sequence to a methionine reduced the activities of both IDH kinase and IDH phosphatase by factors of greater than 500, to levels below the detection limits of the assays. The apparent elimination of both IDH kinase and IDH phosphatase by this mutation is consistent with the proposal that these activities share a common ATP binding site and that these reactions may occur at the same active site. Although conversion of Lys336 to a methionine eliminated detectable IDH kinase activity as measured in vitro, the mutant allele retained the ability to complement an aceK deletion mutation, restoring the ability of these cells to grow on minimal acetate medium. Complementation apparently resulted because the mutant protein retained sufficient activity to phosphorylate IDH in vivo. To determine whether the enzymatic assays performed in vitro had correctly reflected the activity of the mutant protein in vivo, we measured the rates at which mutant and wild-type cultures could incorporate [32P]inorganic phosphate into IDH. The wild-type culture achieved maximal incorporation in less than 3 min. In contrast, 32P incorporation was only barely detectable after 30 min in the mutant culture, indicating that the activity of the mutant protein is, indeed, greatly reduced in vivo. The ability of the mutant allele to complement an aceK null mutation thus suggests that IDH kinase/phosphatase levels in wild-type cells are in great excess over what is required for steady-state growth on acetate medium.     

Isocitrate dehydrogenase mutations: new opportunities for translational research

Over the last decade, comprehensive genome-wide sequencing studies have enabled us to find out unexpected genetic alterations of metabolism in cancer. An example is the identification of arginine missense mutations of isocitrate dehydrogenases-1 and -2 (IDH1/2) in glioma, acute myeloid leukemia (AML), chondrosarcomas, and cholangiocarcinoma. These alterations are closely associated with the production of a new stereospecific metabolite, (R)-2-hydroxyglutarate (R-2HG). A large number of follow-up studies have been performed to address the molecular mechanisms of IDH1/2 mutations underlying how these events contribute to malignant transformation. In the meanwhile, the development of selective mutant IDH1/2 chemical inhibitors is being actively pursued in the scientific community and pharmaceutical industry. The present review article briefly discusses the important findings that highlight the molecular mechanisms of IDH1/2 mutations in cancer and provides a current status for development of selective mutant IDH1/2 chemical inhibitors. [BMB Reports 2015; 48(5): 266-270]     

A single gene codes for the kinase and phosphatase which regulate isocitrate dehydrogenase

The gene which codes for isocitrate dehydrogenase kinase/phosphatase of Escherichia coli, aceK, has been cloned. Physical and functional mapping of this clone indicated that both the isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase activities are encoded by an 1800-base pair sequence. This sequence produced a polypeptide with an apparent molecular weight of 66,000, which is identical to that of the purified protein. Since a protein of this size would require an 1800-base pair coding sequence, we conclude that isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase are expressed from a single gene. This strongly suggests that both activities reside on the same polypeptide chain. The cloning of aceK was made possible by the fortuitous addition of a second origin of replication to the expression vectors which were employed. These expression vectors were found to inhibit the growth of E. coli on the minimal acetate selective medium. The inclusion of a second origin of replication reduced the copy number and so reduced the inhibitory effects of these vectors. Control of the copy number through the addition of replication origins may have a general facility when manipulating plasmids which are potentially toxic to E. coli.