title: Structural analysis and detection of biological inositol pyrophosphates reveal that the family of VIP/diphosphoinositol pentakisphosphate kinases are 1/3-kinases.
abstract: We have characterized the positional specificity of the mammalian and yeast VIP/diphosphoinositol pentakisphosphate kinase (PPIP5K) family of inositol phosphate kinases. We deployed a microscale metal dye detection protocol coupled to a high performance liquid chromatography system that was calibrated with synthetic and biologically synthesized standards of inositol pyrophosphates. In addition, we have directly analyzed the structures of biological inositol pyrophosphates using two-dimensional 1H-1H and 1H-31P nuclear magnetic resonance spectroscopy. Using these tools, we have determined that the mammalian and yeast VIP/PPIP5K family phosphorylates the 1/3-position of the inositol ring in vitro and in vivo. For example, the VIP/PPIP5K enzymes convert inositol hexakisphosphate to 1/3-diphosphoinositol pentakisphosphate. The latter compound has not previously been identified in any organism. We have also unequivocally determined that 1/3,5-(PP)2-IP4 is the isomeric structure of the bis-diphosphoinositol tetrakisphosphate that is synthesized by yeasts and mammals, through a collaboration between the inositol hexakisphosphate kinase and VIP/PPIP5K enzymes. These data uncover phylogenetic variability within the crown taxa in the structures of inositol pyrophosphates. For example, in the Dictyostelids, the major bis-diphosphoinositol tetrakisphosphate is 5,6-(PP)2-IP4 ( Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., Falck, J. R., and Vogel, G. (1996) Biochem. J. 315, 715-725 ). Our study brings us closer to the goal of understanding the structure/function relationships that control specificity in the synthesis and biological actions of inositol pyrophosphates.
authors.affiliation: Institut für Biochemie und Molekularbiologie I: Zelluläre Signaltransduktion, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany.
authors.forename: Hongying
authors.initials: H
authors.lastname: Lin
authors.forename: Peter C
authors.initials: PC
authors.lastname: Fridy
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Ribeiro
authors.forename: Jae H
authors.initials: JH
authors.lastname: Choi
authors.forename: Deb K
authors.initials: DK
authors.lastname: Barma
authors.forename: Günter
authors.initials: G
authors.lastname: Vogel
authors.forename: J R
authors.initials: JR
authors.lastname: Falck
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Shears
authors.forename: John D
authors.initials: JD
authors.lastname: York
authors.forename: Georg W
authors.initials: GW
authors.lastname: Mayr
mesh_terms: D000818:Animals; D016837:Dictyosteliida / Q000201:enzymology; D006801:Humans; D007295:Inositol Phosphates / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D015394:Molecular Structure; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry* / Q000378:metabolism; D010802:Phylogeny; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity / Q000502:physiology; D041701:Swiss 3T3 Cells
chemical_list: D007295:Inositol Phosphates; D015800:Protozoan Proteins; D029701:Saccharomyces cerevisiae Proteins; C079849:1-diphosphoinositol pentakisphosphate; D017856:Phosphotransferases (Phosphate Group Acceptor); C407349:diphosphoinositol pentakisphosphate kinase