Publications

• Isocitrate dehydrogenase kinase/phosphatase. Kinetic characteristics of the wild-type and two mutant proteins.

  Miller S.P., Karschnia E.J., Ikeda T.P., LaPorte D.C.

  Isocitrate dehydrogenase (IDH) of Escherichia coli is regulated by a bifunctional protein, IDH kinase/phosphatase. In addition to the kinase and phosphatase activities, this protein catalyzes an intrinsic ATPase reaction. The initial velocity kinetics of these activities exhibited extensive simila ... >> More

  Isocitrate dehydrogenase (IDH) of Escherichia coli is regulated by a bifunctional protein, IDH kinase/phosphatase. In addition to the kinase and phosphatase activities, this protein catalyzes an intrinsic ATPase reaction. The initial velocity kinetics of these activities exhibited extensive similarities. IDH kinase and phosphatase both yielded intersecting double-reciprocal plots. In addition, we observed similar values for the kinetic constants describing interactions of the kinase and phosphatase with their protein substrates and the interactions of all three activities with ATP. In contrast, while the maximum velocities of IDH kinase and IDH phosphatase were nearly equal, they were 10-fold less than the maximum velocity of the ATPase. Although the IDH phosphatase reaction required either ATP or ADP, it was not supported by the nonhydrolyzable ATP analogue 5'-adenylyl imidodiphosphate. The kinetic properties of wild-type IDH kinase/phosphatase were compared with those of two mutant derivatives of this protein. The mutations in these proteins selectively inhibit IDH phosphatase activity. Inhibition of IDH phosphatase resulted from three factors: decreases in the maximum velocities, reduced affinities for phospho-IDH, and a loss of coupling between ATP and phospho-IDH. These mutations also affected the properties of IDH kinase, increasing the maximum velocities and decreasing the affinities for ATP and phospho-IDH. The intrinsic ATPase activities also exhibited reduced affinity for ATP. These results are discussed in the context of a model which proposes that all three activities occur at the same active site. << Less

  J Biol Chem 271:19124-19128(1996) [PubMed] [EuropePMC]

• Crystal structure of Bacillus subtilis isocitrate dehydrogenase at 1.55 A. Insights into the nature of substrate specificity exhibited by Escherichia coli isocitrate dehydrogenase kinase/phosphatase.

  Singh S.K., Matsuno K., LaPorte D.C., Banaszak L.J.

  Isocitrate dehydrogenase from Bacillus subtilis (BsIDH) is a member of a family of metal-dependent decarboxylating dehydrogenases. Its crystal structure was solved to 1.55 A and detailed comparisons with the homologue from Escherichia coli (EcIDH), the founding member of this family, were made. Al ... >> More

  Isocitrate dehydrogenase from Bacillus subtilis (BsIDH) is a member of a family of metal-dependent decarboxylating dehydrogenases. Its crystal structure was solved to 1.55 A and detailed comparisons with the homologue from Escherichia coli (EcIDH), the founding member of this family, were made. Although the two IDHs are structurally similar, there are three notable differences between them. First, a mostly nonpolar beta-strand and two connecting loops in the small domain of EcIDH are replaced by two polar alpha-helices in BsIDH. Because of a 13-residue insert in this region of BsIDH, these helices protrude over the active site cleft of the opposing monomer. Second, a coil leading into this cleft, the so-called "phosphorylation" loop, is bent inward in the B. subtilis enzyme, narrowing the entrance to the active site from about 12 to 4 A. Third, although BsIDH is a homodimer, the two unique crystallographic subunits of BsIDH are not structurally identical. The two monomers appear to differ by a domain shift of the large domain relative to the small domain/clasp region, reminiscent of what has been observed in the open/closed conformations of EcIDH. In Escherichia coli, IDH is regulated by reversible phosphorylation by the bifunctional enzyme IDH kinase/phosphatase (IDH-K/P). The site of phosphorylation is Ser(113), which lies deep within the active site crevice. Structural differences between EcIDH and BsIDH may explain disparities in their abilities to act as substrates for IDH-K/P. << Less

  J. Biol. Chem. 276:26154-26163(2001) [PubMed] [EuropePMC]

• Structure of the bifunctional isocitrate dehydrogenase kinase/phosphatase.

  Zheng J., Jia Z.

  The Escherichia coli isocitrate dehydrogenase kinase/phosphatase (AceK) is a unique bifunctional enzyme that phosphorylates or dephosphorylates isocitrate dehydrogenase (ICDH) in response to environmental changes, resulting in the inactivation or, respectively, activation of ICDH. ICDH inactivatio ... >> More

  The Escherichia coli isocitrate dehydrogenase kinase/phosphatase (AceK) is a unique bifunctional enzyme that phosphorylates or dephosphorylates isocitrate dehydrogenase (ICDH) in response to environmental changes, resulting in the inactivation or, respectively, activation of ICDH. ICDH inactivation short-circuits the Krebs cycle by enabling the glyoxlate bypass. It was the discovery of AceK and ICDH that established the existence of protein phosphorylation regulation in prokaryotes. As a 65-kDa protein, AceK is significantly larger than typical eukaryotic protein kinases. Apart from the ATP-binding motif, AceK does not share sequence homology with any eukaryotic protein kinase or phosphatase. Most intriguingly, AceK possesses the two opposing activities of protein kinase and phosphatase within one protein, and specifically recognizes only intact ICDH. Additionally, AceK has strong ATPase activity. It has been shown that AceK kinase, phosphatase and ATPase activities reside at the same site, although the molecular basis of such multifunctionality and its regulation remains completely unknown. Here we report the structures of AceK and its complex with ICDH. The AceK structure reveals a eukaryotic protein-kinase-like domain containing ATP and a regulatory domain with a novel fold. As an AceK phosphatase activator and kinase inhibitor, AMP is found to bind in an allosteric site between the two AceK domains. An AMP-mediated conformational change exposes and shields ATP, acting as a switch between AceK kinase and phosphatase activities, and ICDH-binding induces further conformational change for AceK activation. The substrate recognition loop of AceK binds to the ICDH dimer, allowing higher-order substrate recognition and interaction, and inducing critical conformational change at the phosphorylation site of ICDH. << Less

  Nature 465:961-965(2010) [PubMed] [EuropePMC]

• The 'catalytic' triad of isocitrate dehydrogenase kinase/phosphatase from E. coli and its relationship with that found in eukaryotic protein kinases.

  Oudot C., Cortay J.-C., Blanchet C., Laporte D.C., Di Pietro A., Cozzone A.J., Jault J.-M.

  The isocitrate dehydrogenase kinase/phosphatase (IDHK/P) of E. coli is a bifunctional enzyme responsible for the reversible phosphorylation of isocitrate dehydrogenase (IDH) on a seryl residue. As such, it belongs to the serine/threonine protein kinase family. However, only a very limited homology ... >> More

  The isocitrate dehydrogenase kinase/phosphatase (IDHK/P) of E. coli is a bifunctional enzyme responsible for the reversible phosphorylation of isocitrate dehydrogenase (IDH) on a seryl residue. As such, it belongs to the serine/threonine protein kinase family. However, only a very limited homology with the well-characterized eukaryotic members of that family was identified so far in its primary structure. In this report, a new region of amino acids including three putative residues involved in the kinase activity of IDHK/P was identified by sequence comparison with eukaryotic protein kinases. In IDHK/P, these residues are Asp-371, Asn-377, and Asp-403. Their counterpart eukaryotic residues have been shown to be involved in either catalysis (former residue) or magnesium binding (the two latter residues). Site-directed mutagenesis was performed on these three IDHK/P residues, and also on the Glu-439 residue equivalent to that of the Ala-Pro-Glu motif found in the eukaryotic protein kinases. Mutations of Asp-371 into either Ala, Glu, or Gln residues drastically lowered the yield and the quality of the purification. Nevertheless, the recovered mutant enzymes were barely able to phosphorylate IDH either in vitro or after expression in an aceK (-) mutant strain. In contrast, mutation of either Asn-377, Asp-403, or Glu-439 into an Ala residue altered neither the yield of purification nor the maximal phosphorylating capacity of the enzyme. However, when IDH was phosphorylated in the presence of increasing concentrations of magnesium ions, the two former mutants displayed a much lower affinity for this cation, with a K(m) value of 0.6 or 0.8 mM, respectively, as compared to 0.1 mM for the wild-type enzyme. On the other hand, the Glu439Ala mutant has an affinity for magnesium essentially unaffected. Therefore, and in contrast to the current opinion, our results suggest that the catalytic mechanism of IDHK/P exhibits some similarities with that found in the eukaryotic members of the protein kinase family. << Less

  Biochemistry 40:3047-3055(2001) [PubMed] [EuropePMC]