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Literature summary extracted from

  • Wang, H.; Shears, S.B.
    Structural features of human inositol phosphate multikinase rationalize its inositol phosphate kinase and phosphoinositide 3-kinase activities (2017), J. Biol. Chem., 292, 18192-18202 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.1.151 gene ipmk, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli Homo sapiens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.7.1.151 purified recombinant core catalytic domain of HsIMPK, that contains residues 50 to 416, from which an internal domain comprising residues 263 to 377 is deleted and replaced with a simple Gly-Gly-Ser-Gly-Gly linker, apostructure and catalytic core domain with bound ADP-Ins (1,4,5)P3-Mg and ADP-DiC4-PtdInsP2-Mg, hanging drop vapor diffusion, mixing of 0.002 ml of 38 mg/ml protein solution with 0.002 ml of well solution containing 35% w/v PEG 400, 0.1 M Li2SO4, 100 mM MES-imidazole, pH 6.0, and 50 mM 2-mercaptoethanol, 25°C, to obtain complex structures, apoenzyme crystals are further soaked for 1 day in 35% w/v PEG 400, 100 mM Li2SO4, 100 mM HEPES, pH 7.5, at 25°C, in the presence of 20 mM Ins(1,4,5)P3 or a soluble diC4-analogue of PtdIns(4,5)P2, 10 mM MgCl2, and 5 mM of either Na2ATP or Li2AMP-PNP, X-ray diffraction structure determination and analysis at 1.63-1.93 A resolution. The structure of the IPMK apoenzyme is determined by a molecular replacement approach using a model constructed from the template of yeast ScIPMK (PDB ID 2IF8) Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
2.7.1.151 H388A site-directed mutagenesis, the mutant shows no Ins(1,4,5)P3 3-kinase activity and reduced Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens
2.7.1.151 K160A site-directed mutagenesis, the mutant shows no Ins(1,4,5)P3 3-kinase activity and reduced Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens
2.7.1.151 K167A site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens
2.7.1.151 additional information generation of the core catalytic domain of the enzyme that contains residues 50 to 416, from which an internal domain comprising residues 263 to 377 is deleted. The deletion is necessary to obtain crystals, it is replaced with a simple Gly-Gly-Ser-Gly-Gly linker. This deletion does not compromise catalytic activity, it is a non-catalytic region of the protein. It contains a nuclear localization sequence, flanked by residues that host protein kinase phosphorylation sites that regulate nuclear localization sequence functionality. Gln residues at positions 163, 164, and 196 are mutated each to Arg and Lys, both of which have side chains that are larger and also positively charged at physiological pH. The results are quite dramatic: in each case, the rate of Ins(1,4,5)P3 3-kinase activity declines, but in contrast, the rate of Ins(1,3,4,5)P4 6-kinase activity is not impaired, three of these mutants show increased 6-kinase activity, analysis of the structural basis, overview Homo sapiens
2.7.1.151 Q163A site-directed mutagenesis, the mutant shows no Ins(1,4,5)P3 3-kinase activity and reduced Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens
2.7.1.151 Q163K site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and increased Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens
2.7.1.151 Q163R site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and increased Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens
2.7.1.151 Q164A site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens
2.7.1.151 Q164K site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and increased Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens
2.7.1.151 Q164R site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and increased Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens
2.7.1.151 Q196A site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens
2.7.1.151 Q196K site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and increased Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens
2.7.1.151 Q196R site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and increased Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens
2.7.1.151 Q78A site-directed mutagenesis, the mutant shows no Ins(1,4,5)P3 3-kinase activity and reduced Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens
2.7.1.151 R82A site-directed mutagenesis, the mutant shows no Ins(1,4,5)P3 3-kinase activity and reduced Ins(1,3,4,5)P4 6-kinase activity compared to wild-type Homo sapiens

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
2.7.1.151 nucleus the internal domain comprising residues 263 to 377 contains the nuclear localization sequence Homo sapiens 5634
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.1.151 Mg2+ required Homo sapiens

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.1.151 ATP + 1D-myo-inositol 1,3,4,5-tetrakisphosphate Homo sapiens
-
 ADP + 1D-myo-inositol 1,3,4,5,6-pentakisphosphate
-
 ?
2.7.1.151 ATP + 1D-myo-inositol 1,4,5-trisphosphate Homo sapiens
-
 ADP + 1D-myo-inositol 1,3,4,5-tetrakisphosphate
-
 ?
2.7.1.151 ATP + 1D-myo-inositol 4,5-bisphosphate Homo sapiens
-
 ADP + 1D-myo-inositol 3,4,5-trisphosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.7.1.151 Homo sapiens Q8NFU5
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.1.151 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity and heparin affinity chromatography, followed by tag cleavage through TEV protease, another step of heparin affinity chromatography, and gel filtration Homo sapiens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.1.151 ATP + 1D-myo-inositol 1,3,4,5-tetrakisphosphate
-
 Homo sapiens ADP + 1D-myo-inositol 1,3,4,5,6-pentakisphosphate
-
 ?
2.7.1.151 ATP + 1D-myo-inositol 1,4,5-trisphosphate
-
 Homo sapiens ADP + 1D-myo-inositol 1,3,4,5-tetrakisphosphate
-
 ?
2.7.1.151 ATP + 1D-myo-inositol 4,5-bisphosphate
-
 Homo sapiens ADP + 1D-myo-inositol 3,4,5-trisphosphate
-
 ?
2.7.1.151 additional information recombinant HsIMPK uses 3-kinase activity to phosphorylate Ins(1,4,5)P3 to Ins(1,3,4,5)P4, and then phosphorylates the latter with a 6-kinase activity that yields Ins(1,3,4,5,6)P5. It also shows PtdIns(4,5)P2 3-kinase activity. Substrate binding structures, overview Homo sapiens ?
-
-

Synonyms

EC Number Synonyms Comment Organism
2.7.1.151 HsIPMK
-
 Homo sapiens
2.7.1.151 inositol phosphate multikinase
-
 Homo sapiens
2.7.1.151 IP3K
-
 Homo sapiens
2.7.1.151 IPMK
-
 Homo sapiens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.7.1.151 37
-
 assay at Homo sapiens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.7.1.151 7.2
-
 assay at Homo sapiens

Cofactor

EC Number Cofactor Comment Organism Structure
2.7.1.151 ATP
-
 Homo sapiens

General Information

EC Number General Information Comment Organism
2.7.1.151 evolution IPMK is a member of the so-called IP-kinase family that includes IP3Ks and IP6Ks, evolutionary relationships and structure comparisons, overview. There has been co-evolution of Ins(1,4,5)P3 and PtdIns(4,5)P2 3-kinase activities Homo sapiens
2.7.1.151 additional information HsIPMK owns a catalytic pocket that is more constrained than those of the plant and yeast orthologues. Also unique to mammalian IPMK is a catalytically important proline-loop, and a preponderance of Gln residues in the active site. Description of two versions of Ins(1,4,5)P3 within the active site, first as a free inositol phosphate, and second as the headgroup of a soluble analogue of PtdIns(4,5)P2. The structure of the IPMK apoenzyme is determined by a molecular replacement approach using a model constructed from the template of yeast ScIPMK (PDB accession code 2IF8), and this apo-structure is used for further elucidation of the structures of crystal complexes with ADP plus either Ins(1,4,5)P3 or diC4-PtdIns(4,5)P2. Domains that are similar to the so-called N- and C-lobes that comprise the ATP-binding site. The C-terminal lobe comprising residues 136-149 and 175-416, which is an alphabeta-fold with five, central antiparallel beta-strands including beta4-6, beta8, and beta9, a pair of small antiparallel beta-strands (beta7 and beta10), and three alpha-helices (alpha5-alpha7). Also in the C-lobe of HsIPMK, a 310 helix is observed between the beta6 strand and alpha5 helix. His388 is at the catalytic center. Structure comparisons, overview Homo sapiens
2.7.1.151 physiological function human inositol phosphate multikinase (HsIPMK) critically contributes to intracellular signaling through its inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) 3-kinase activities. HsIPMK is both an inositol phosphate kinase and a PtdIns(4,5)P2 kinase Homo sapiens