Literature summary for 2.7.4.21 extracted from

Wang, H.; DeRose, E.F.; London, R.E.; Shears, S.B.
IP6K structure and the molecular determinants of catalytic specificity in an inositol phosphate kinase family (2014), Nat. Commun., 5, 4178.

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of MBP-tagged EhIP6KA residues 32-270	Entamoeba histolytica

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant MBP-tagged EhIP6KA residues 32-270, several crystal complexes of the IP6K including those that contain either large (Ins(1,3,4,5,6)P5 /InsP6) or small (Ins(1,4,5)P3) substrates, hanging drop vapor diffusion, 60 mg/ml protein in solution is mixed with reservoir solution containing 12% w/v PEG 3350, 50 mM NaH2PO4, pH 5.5 at 18°C, or by hanging drop vapor diffusion for one week against a well buffer containing 8% w/v PEG 3350, 100 mM Na3citrate, pH 5.2 at 25°C, followed by dilution microseeding for two weeks with a well buffer of 8% w/v PEG 3350, 100 mM Na3citrate, pH 5.2, and 8% ethylene glycol at 25°C, soaking of crystals in 22% w/v PEG 3350, 10 mM MgCl2, 10 mM ATP, 0.1 M sodium acetate, pH 5.2, and 20 mM InsP6, 10 mM Ins(1,3,4,5,6)P5 or 10 mM Ins(1,4,5)P3 for 3 days, X-ray diffraction structure determination and analysis, molecular replacement using MBP (1ez9) and IP3K (1w2c) as search models	Entamoeba histolytica

Crystallization (Comment)

Organism

purified recombinant MBP-tagged EhIP6KA residues 32-270, several crystal complexes of the IP6K including those that contain either large (Ins(1,3,4,5,6)P5 /InsP6) or small (Ins(1,4,5)P3) substrates, hanging drop vapor diffusion, 60 mg/ml protein in solution is mixed with reservoir solution containing 12% w/v PEG 3350, 50 mM NaH2PO4, pH 5.5 at 18°C, or by hanging drop vapor diffusion for one week against a well buffer containing 8% w/v PEG 3350, 100 mM Na3citrate, pH 5.2 at 25°C, followed by dilution microseeding for two weeks with a well buffer of 8% w/v PEG 3350, 100 mM Na3citrate, pH 5.2, and 8% ethylene glycol at 25°C, soaking of crystals in 22% w/v PEG 3350, 10 mM MgCl2, 10 mM ATP, 0.1 M sodium acetate, pH 5.2, and 20 mM InsP6, 10 mM Ins(1,3,4,5,6)P5 or 10 mM Ins(1,4,5)P3 for 3 days, X-ray diffraction structure determination and analysis, molecular replacement using MBP (1ez9) and IP3K (1w2c) as search models

Entamoeba histolytica

Protein Variants

Protein Variants	Comment	Organism
additional information	construction of an Entamoeba histolytica hybrid IP6K/IP3K chimeric hybrid through molecular modeling and mutagenesis	Entamoeba histolytica

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Entamoeba histolytica

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + 1D-myo-inositol hexakisphosphate	Entamoeba histolytica	-	ADP + 1D-myo-inositol 5-diphosphate 1,2,3,4,6-pentakisphosphate	-	?
additional information	Entamoeba histolytica	secondary to its InsP6 kinase activity, also phosphorylates the 6-OH of Ins(1,4,5)P3, an inositol phosphate multikinase (IPMK)-like activity. IPMK itself is positionally promiscuous in that it is a 3-, 5- and 6-kinase. The enzyme also shows significant InsP3 kinase activity	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Entamoeba histolytica	C4M387	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + 1D-myo-inositol hexakisphosphate	-	Entamoeba histolytica	ADP + 1D-myo-inositol 5-diphosphate 1,2,3,4,6-pentakisphosphate	-	?
additional information	secondary to its InsP6 kinase activity, also phosphorylates the 6-OH of Ins(1,4,5)P3, an inositol phosphate multikinase (IPMK)-like activity. IPMK itself is positionally promiscuous in that it is a 3-, 5- and 6-kinase. The enzyme also shows significant InsP3 kinase activity	Entamoeba histolytica	?	-	?
additional information	EhIP6KA, like mammalian IP6Ks, converts InsP6 to 5-InsP7. The 5-phosphate group on InsP6 is diphosphorylated by EhIP6KA. The enzyme phosphorylates Ins(1,3,4,5,6)P5, and the major product co-elutes with a PP-[3H]InsP4 standard, no InsP6 is formed. The first order rate constant for Ins(1,3,4,5,6)P5 phosphorylation is 40fold lower than that for InsP6. The rate of phosphorylation of Ins(1,4,5)P3, is 220fold slower than that for InsP6, either the 2- or the 6-OH of Ins(1,4,5)P3 are phosphorylated. Ins(1,4,5,6)P4 is the major InsP4 product formed from Ins(1,4,5)P, with phosphorylation of an inositol phosphate at the 2-position. Steric restrictions prevent InsP6 from occupying the same space as Ins(1,4,5)P3 in the substrate-binding pocket. Substrate binding and specificities, overview	Entamoeba histolytica	?	-	?

Subunits

Subunits	Comment	Organism
monomer	gel filtration and sequence calculation	Entamoeba histolytica
More	enzyme structure determinaion and comparisons, similarities to enzymes human IP3KA, human IP6K, and Saccharomyces cerevisiae IPMK, overview	Entamoeba histolytica

Synonyms

Synonyms	Comment	Organism
EhIP6KA	-	Entamoeba histolytica
IP6K	-	Entamoeba histolytica

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Entamoeba histolytica

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Entamoeba histolytica

Cofactor

Cofactor	Comment	Organism	Structure
ATP	binding site structure, overview	Entamoeba histolytica

General Information

General Information	Comment	Organism
evolution	IP6Ks are members of a wider inositol phosphate kinase family (Pfam PF03770) that includes IPMKs and IP3Ks. These enzymes all share a PxxxDxKxG, PDKG, catalytic motif. Phylogenetic analysis indivates that this kinase family arose from a primordial IP6K precursor	Entamoeba histolytica
additional information	structural analysis of the IP6K from Entamoeba histolytica	Entamoeba histolytica