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Characterization of A. thaliana adenylate - isopentenyltransferases

Characterization of A. thaliana adenylate - isopentenyltransferases. Markéta Gemrotová. Isopentenyltransferases (IPT)?. Biosynthesis of isoprenoid-type cytokinins Two types of IPT enzymes: adenylate-isopentenyltransferase ( At IPT1,3,4,5,7,8 ):

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Characterization of A. thaliana adenylate - isopentenyltransferases

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  1. Characterizationof A.thalianaadenylate-isopentenyltransferases Markéta Gemrotová

  2. Isopentenyltransferases (IPT)? • Biosynthesis of isoprenoid-type cytokinins • Two types of IPT enzymes: adenylate-isopentenyltransferase(AtIPT1,3,4,5,7,8): tRNA-isopentenyltransferase: tRNA modification (AtIPT2,AtIPT9) Localization: AtIPT1,5,8 plastids AtIPT7 mitochondria AtIPT2,9 a 4 v cytosol AtIPT3 farnesylated in nucleus/cytoplasm AtIPT3 plastids Gene s for IPT found in bacteria (A.tumefaciens, R.fascians…), yeasts S. cerevisisiae, tRNA-IPT even in human DNA.

  3. Previously published data? • AtIPT1 Takei et al.(2001), AtIPT4 Kakimoto(2001) ….Missing kinetic data for other substrates  Miyawaki et al.(2006) tRNA-IPT mutants of A.thaliana had low cisZ – evidence that cisZ arises only from prenyled-tRNA? Biochemically not confirmed… • Galichet et al. (2008)- AtIPT3 is modificated- farnesylation dependent translocation and activity !! AtIPT7 has also farnesylation site… • Low enzyme activity,stabilityand weak expression discouraged deeper exploration 

  4. Charakterization of AtIPT • RecombinantAtIPT 1, 4, 5 and 8 fromE. coli • Kinetic measurements for various combinations of substrates AMP/ADP/ATP andisoprenylprecoursors, determinationof Km, kcat : Dimethylallyldiphosphate: Isopentenyladenineprecursor (2E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate : transZ precursor (2Z)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate :cisZ prekurzor ?

  5. Purification of AtIPT1, AtIPT4, AtIPT5, AtIPT8 IPT1 IPT4 IPT8 IPT5

  6. AtIPT1 AtIPT4 AtIPT5 AtIPT8

  7. AtIPT1 AtIPT4 AtIPT5 AtIPT8

  8. Site-directed mutagenesis AtIPT1 E220L (glutamic acid » leucin), AtIPT4 R157A (arginin » alanin),  AtIPT4 R157D (arginin » aspartic acid), AtIPT4 R157E (arginin » glutamic acid) AtIPT4 L160E (lysin » glutamic acid), AtIPT4 L160D (lysin » aspartic acid), • AtIPT4 N50G (asparagin » » » glycin) • AtIPT4 N50S (asparagin » » » serin) mut8 mut9

  9. AtIPT4

  10. cHMBDP +ATP in Ser mutant cHMBDP +ATP in Gly mutant

  11. Light at the end of the tunnel ? • Characterization of AtIPT3 Expression of IPT3 in S. cerevisiae to get farnesylated protein. In progress

  12. Acknowledgements: Laboratory of Growth Regulators,Palacký University & Institute of Experimental Botany AS CR: Lukáš Spíchal Václav Mik Miroslav Strnad Leibniz-Institute of Plant Biochemistry, Department of Bio-Organic Chemistry, Halle: Dimitar Vasilev Robert Klein Ludger Wessjohann Department of Biochemistry-Laboratory of Molecular Biology, Faculty of Science, Palacký University in Olomouc: Mária Šmehilová and Petr Galuszka GA CR 522/08/0920 andMSM 6198959216

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