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 kinase/phosphatase

In Escherichia coli, isocitrate dehydrogenase (IDH) is regulated by phosphorylation. This phosphorylation cycle is catalyzed by an unusual, bifunctional protein:IDH kinase/phosphatase. IDH kinase/phosphatase is expressed from a single gene, aceK, and both activities are catalyzed by the same polypeptide. The amino acid sequence of IDH kinase/phosphatase does not exhibit the characteristics which are typical of other protein kinases, although it does contain a consensus ATP binding site. The available evidence suggests that the IDH kinase and IDH phosphatase reactions occur at the same active site and that the IDH phosphatase reaction results from the back reaction of IDH kinase tightly coupled to ATP hydrolysis. The function of the IDH phosphorylation cycle is to control the flux of isocitrate through the glyoxylate bypass. This pathway is essential for growth on acetate because it prevents the quantitative loss of the acetate carbons as CO2 in the Krebs' cycle. IDH kinase/phosphatase monitors general metabolism by responding to the levels of a wide variety of metabolites, many of which activate IDH phosphatase and inhibit IDH kinase. The ability of IDH kinase/phosphatase to monitor general metabolism allows. the IDH phosphorylation cycle to compensate for substantial perturbations of the system, such as a 15-fold overproduction of IDH. The significance of the cellular level of IDH kinase/phosphatase has also been evaluated. The level of this protein is in great excess of that required for steady-state growth on acetate. In contrast, IDH kinase/phosphatase is, in some cases, rate-limiting for the dephosphorylation of IDH which results when preferred carbon sources are added to cultures growing on acetate.     

Size and Sequence Polymorphism in the Isocitrate Dehydrogenase Kinase/Phosphatase Gene (Acek) and Flanking Regions in Salmonella Enterica and Escherichia Coli

The sequence of aceK, which codes for the regulatory catalytic enzyme isocitrate dehydrogenase kinase/phosphatase (IDH K/P), and sequences of the 5' flanking region and part or all of the 3' flanking region were determined for 32 strains of Salmonella enterica and Escherichia coli. In E. coli, the aceK gene was 1734 bp long in 13 strains, but in three strains it was 12 bp shorter and the stop codon was TAA rather than TGA. Strains with the shorter aceK lacked an open reading frame (f728) downstream between aceK and iclR that was present, in variable length, in the other strains. Among the 72 ECOR strains, the truncated aceK gene was present in all isolates of the B2 group and half of those of the D group. Other variant conditions included the presence of IS1 elements in two strains and large deletions in two strains. The aceK-aceA intergenic region varied in length from 48 to 280 bp in E. coli, depending largely on the number of repetitive extragenic palindromic (REP) sequences present. Among the ECOR strains, the number of REP elements showed a high degree of phylogenetic association, and sequencing of the region in the ECOR strains permitted partial reconstruction of its evolutionary history. In S. enterica, the normal length of aceK was 1752 bp, but three other length variants, ranging from 1746 to 1785 bp, were represented in five of the 16 strains examined. The flanking intergenic regions showed relatively minor variation in length and sequence. The occurrence of several nonrandom patterns of distribution of polymorphic synonymous nucleotide sites indicated that intragenic recombination of horizontally exchanged DNA has contributed to the generation of allelic diversity at the aceK locus in both species.     

Glyoxylate bypass operon of Escherichia coli: cloning and determination of the functional map.

In Escherichia coli, a single operon encodes the metabolic and regulatory enzymes of the glyoxylate bypass. The metabolic enzymes, isocitrate lyase and malate synthase, are expressed from aceA and aceB, and the regulatory enzyme, isocitrate dehydrogenase kinase/phosphatase, is expressed from aceK. We cloned this operon and determined its functional map by deletion analysis. The order of the genes in this operon is aceB-aceA-aceK, with aceB proximal to the promoter, consistent with the results of previous experiments using genetic techniques. The promoter was identified by S1 nuclease mapping, and its nucleotide sequence was determined. Isocitrate lyase and malate synthase were readily identified by autoradiography after the products of the operon clone were labeled by the maxicell procedure and then resolved by electrophoresis. In contrast, isocitrate dehydrogenase kinase/phosphatase, expressed from the same plasmid, was undetectable. This observation is consistent with a striking downshift in expression between aceA and aceK.     

Compensatory phosphorylation of isocitrate dehydrogenase. A mechanism for adaptation to the intracellular environment

When Escherichia coli grows on acetate, the flow of isocitrate through the glyoxylate bypass is regulated, in part, through the phosphorylation of isocitrate dehydrogenase. In addition to its role in adaptation to alternative carbon sources, this phosphorylation system responds to variation in the intracellular level of isocitrate dehydrogenase. This system can compensate for changes in the cellular level of isocitrate dehydrogenase in excess of 10-fold, maintaining a nearly constant activity for isocitrate dehydrogenase during growth on acetate. The behavior of the phosphorylation system exhibited considerable strain-specific variation. This was most clearly demonstrated using mutants which lacked the ability to phosphorylate isocitrate dehydrogenase. In two strains, mutation of the gene for isocitrate dehydrogenase kinase/phosphatase rendered the cells unable to grow on acetate. In contrast, a third strain was relatively insensitive to a mutation in this gene. This lack of phenotypic expression appears to result from a lower cellular level of isocitrate dehydrogenase in this strain which renders the phosphorylation (and consequent inhibition) of isocitrate dehydrogenase less essential. The gene for isocitrate dehydrogenase kinase/phosphatase (aceK) was located in the glyoxylate bypass operon, downstream from the genes for isocitrate lyase and malate synthase.     

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 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.     

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.     

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.     

The amino acid sequence of sarcoplasmic calcium-binding protein obtained from sandworm, Perinereis vancaurica tetradentata

Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1000-fold from Escherichia coli ML308 by procedure involving fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose, blue-dextran-Sepharose and Sephadex G150. The kinase and phosphatase activities copurified, in agreement with the observation [Laporte, D.C. and Koshland, D.E. (1982) Nature (Lond.) 300, 458-460] that a single protein bears both activities. Isocitrate dehydrogenase kinase catalysed the phosphorylation of homogeneous active isocitrate dehydrogenase with a stoichiometry of just under one phosphate group incorporated per subunit. This almost completely inactivated the dehydrogenase. There was a good correlation between phosphorylation and inactivation. Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine. Isocitrate dehydrogenase phosphatase catalysed the release of 32P from 32P-phosphorylated isocitrate dehydrogenase; it required either ADP or ATP for activity. In the presence of ADP, or ATP plus an inhibitor of the kinase, the phosphatase catalysed full reactivation of isocitrate dehydrogenase and there was a good correlation between reactivation and the release of phosphate. In the presence of ATP alone the phosphatase catalysed the release of 32P from phosphorylated isocitrate dehydrogenase but the activity of the dehydrogenase remained low, indicating that the kinase and phosphatase were active simultaneously in these conditions. The active and inactive forms of isocitrate dehydrogenase can be resolved by non-denaturing gel electrophoresis; the two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in vitro. The extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity.     

TraJ protein of plasmid RP4 binds to a 19-base pair invert sequence repetition within the transfer origin

Transfer of plasmid RP4 during bacterial conjugation requires the plasmid-encoded TraJ protein, which binds to the transfer origin (Fürste, J. P., Pansegrau, W., Ziegelin, G., Kr?ger, M., and Lanka, E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1771-1775). As indicated by traJ mutants, the TraJ protein is a constituent of the relaxosome, the initiation complex of transfer DNA replication. The traJ gene maps adjacent to the transfer origin (oriT). The structural gene consists of a 372-base pair sequence encoding a polypeptide of 122 amino acids (13,282 Da). TraJ was purified from an Escherichia coli strain overproducing the protein. DNA footprinting experiments involving DNase I demonstrated that the purified protein binds to the right arm of a 19-base pair inverted repeat within oriT. Hydroxyl radical footprints of the DNA-protein complex revealed that TraJ protein is bound to only one side of the DNA helix.     

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.     

Control of metabolic interconversion of isocitrate dehydrogenase between the catalytically active and inactive forms in Escherichia coli

The enzymic interconversion of Escherichia coli isocitrate dehydrogenase (ICDH) between the catalytically active and inactive forms is mediated through the activities of ICDH-kinase/phosphatase in response to changes in the metabolic environment. In this study, the use of mutant strains devoid of isocitrate lyase ( aceA:: Tn10 ) and pyruvate dehydrogenase activities revealed that the signal which triggers the reversible inactivation of ICDH in vivo is not directly related to acetate itself, but rather to the need to maintain high intracellular levels of isocitrate and free co-enzyme A. The use of these mutants also revealed, rather unexpectedly, that acetate grown cells contain more ICDH protein than those grown with other carbon sources and that the catalytic activity of ICDH kinase/phosphatase is in excess of cellular demands. Furthermore, this study also revealed the presence of a 50-kDa (±2 kDa) acetate-specific polypeptide, the identity of which has yet to be established.     

Deletion analysis of the DNA sequence required for the in vitro initiation of replication of bacteriophage lambda

Supercoiled DNA containing the replication origin of bacteriophage lambda can be replicated in vitro. This reaction requires purified lambda O and P replication proteins and a partially purified mixture of Escherichia coli proteins (Tsurimoto, T., and Matsubara, K. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7639-7643; Wold, M. S., Mallory, J.B., Roberts, J. D., LeBowitz, J. H., and McMacken, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6176-6180). The lambda origin region has four repeats of a 19-base pair sequence to which O protein binds. To the right of these sites on the lambda map is a 40-base pair region that is rich in adenine and thymine, followed by a 28-base pair palindromic sequence. To define more precisely the boundaries of the lambda origin, we cloned a 358-base pair piece of lambda DNA containing the origin region into M13mp8 in both orientations. In vitro replication of RF I DNAs prepared from cells infected with these two M13 ori lambda phage was dependent on lambda O and P proteins and a crude protein fraction from uninfected E. coli; with these conditions there was no replication of M13mp8 RF I DNA. We made deletions from the left and the right ends of the lambda origin DNA and determined the deletion end points by DNA sequencing. We have tested RF I DNAs prepared from cells infected with phage carrying ori lambda deletions for their ability to function as templates for O- and P-dependent replication in vitro. Our results show that lambda DNA between nucleotide positions 39072 and 39160 is required for efficient O- and P-dependent replication. This 89-base pair piece of DNA includes only two of the four 19-base pair O protein-binding sites (the two right-most) and the adjoining adenine- and thymine-rich region to the right of the O-binding sites.     

Studies of the phosphorylation of Escherichia coli isocitrate dehydrogenase. Recognition of the enzyme by isocitrate dehydrogenase kinase/phosphatase and effects of phosphorylation on its structure and properties

Escherichia coli isocitrate dehydrogenase is completely inactivated by phosphorylation of a single serine residue per subunit. We have examined the conformations of the active and phosphorylated forms of the enzyme using circular dichroism spectroscopy. The results support the view that phosphorylation prevents the binding of NADP, probably by direct blocking of the coenzyme-binding site. Labelling studies suggest that an arginine residue at the coenzyme-binding site may be close to the phosphorylatable serine residue. The phosphorylation of isocitrate dehydrogenase is thus unusual in that it occurs at the active site of the enzyme. We therefore investigated the recognition of isocitrate dehydrogenase by isocitrate dehydrogenase kinase/phosphatase. The kinase activity of this enzyme can phosphorylate intact isocitrate dehydrogenase but not proteolytic fragments derived from it, nor a synthetic peptide corresponding to the sequence round the phosphorylation site.