Biochemical and functional characterization of inositol 1,3,4,5, 6-pentakisphosphate 2-kinases.
Synthesis of inositol 1,2,3,4,5,6-hexakisphosphate (IP(6)), also known as phytate, is integral to cellular function in all eukaryotes. Production of IP(6) predominately occurs through phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (IP(5)) by a 2-kinase. Recent cloning of the gene encoding this kinase from Saccharomyces cerevisiae, designated scIpk1, has identified a cellular role for IP(6) production in the regulation of mRNA export from the nucleus. In this report, we characterize the biochemical and functional parameters of recombinant scIpk1. Purified recombinant scIpk1 kinase activity is highly selective for IP(5) substrate and exhibits apparent K(m) values of 644 nm and 62.8 microm for IP(5) and ATP, respectively. The observed apparent catalytic efficiency (k(cat)/K(m)) of scIpk1 is 31,610 s(-)(1) m(-)(1). A sequence similarity search was used to identify an IP(5) 2-kinase from the fission yeast Schizosaccharomyces pombe. Recombinant spIpk1 has similar substrate selectivity and catalytic efficiency to its budding yeast counterpart, despite sharing only 24% sequence identity. Cells lacking sc-IPK1 are deficient in IP(6) production and exhibit lethality in combination with a gle1 mutant allele. Both of these phenotypes are complemented by expression of the spIPK1 gene in the sc-ipk1 cells. Analysis of several inactive mutants and multiple sequence alignment of scIpk1, spIpk1, and a putative Candida albicans Ipk1 have identified residues involved in catalysis. This includes two conserved motifs: E(i/l/m)KPKWL(t/y) and LXMTLRDV(t/g)(l/c)(f/y)I. Our data suggest that the mechanism for IP(6) production is conserved across species.
Ives
E B
EB
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Nichols
J
J
Wente
S R
SR
York
J D
JD
D000595:Amino Acid Sequence; D002176:Candida albicans; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010833:Phytic Acid / Q000096:biosynthesis; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology; D016415:Sequence Alignment
D029701:Saccharomyces cerevisiae Proteins; D010833:Phytic Acid; C120127:IPK1 protein, S cerevisiae; D017853:Phosphotransferases (Alcohol Group Acceptor)