The synthesis of inositol hexakisphosphate. Characterization of human inositol 1,3,4,5,6-pentakisphosphate 2-kinase.
The enzyme(s) responsible for the production of inositol hexakisphosphate (InsP(6)) in vertebrate cells are unknown. In fungal cells, a 2-kinase designated Ipk1 is responsible for synthesis of InsP(6) by phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (InsP(5)). Based on limited conserved sequence motifs among five Ipk1 proteins from different fungal species, we have identified a human genomic DNA sequence on chromosome 9 that encodes human inositol 1,3,4,5,6-pentakisphosphate 2-kinase (InsP(5) 2-kinase). Recombinant human enzyme was produced in Sf21 cells, purified, and shown to catalyze the synthesis of InsP(6) or phytic acid in vitro. The recombinant protein converted 31 nmol of InsP(5) to InsP(6)/min/mg of protein (V(max)). The Michaelis-Menten constant for InsP(5) was 0.4 microM and for ATP was 21 microM. Saccharomyces cerevisiae lacking IPK1 do not produce InsP(6) and show lethality in combination with a gle1 mutant allele. Here we show that expression of the human InsP(5) 2-kinase in a yeast ipk1 null strain restored the synthesis of InsP(6) and rescued the gle1-2 ipk1-4 lethal phenotype. Northern analysis on human tissues showed expression of the human InsP(5) 2-kinase mRNA predominantly in brain, heart, placenta, and testis. The isolation of the gene responsible for InsP(6) synthesis in mammalian cells will allow for further studies of the InsP(6) signaling functions.
Verbsky
John W
JW
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Wilson
Monita P
MP
Kisseleva
Marina V
MV
Majerus
Philip W
PW
Wente
Susan R
SR
D000595:Amino Acid Sequence; D000818:Animals; D000852:Anopheles / Q000201:enzymology; D017173:Caenorhabditis elegans / Q000201:enzymology; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010833:Phytic Acid / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014714:Vertebrates
D011994:Recombinant Proteins; D010833:Phytic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); C064990:inositol pentakisphosphate kinase