1. 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 - t RNA? Biochemically not confirmed … - Galichet et al. (2008)- AtIPT3 is modificated- farnesylation dependent translocation and activity !! AtIPT7 has also farnesylation site… - Low enzyme activity, stability and weak expression discouraged deeper exploration

4. Charakterization of AtIPT  Recombinant AtIPT 1, 4, 5 and 8 from E. coli  Kinetic measurements for various combinations of substrates AMP/ADP/ATP and isoprenyl precoursors, determination of Km, kcat : (2E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate : transZ precursor (2Z)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate : cisZ prekurzor ? Dimethylallyldiphosphate: Isopentenyladenine precursor

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

6. AtIPT1 AtIPT8 AtIPT4 AtIPT5

7. AMPADPATP AMPADPATP AtIPT1AtIPT4 AtIPT5AtIPT8

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 SubstratesEnzymeK m [ µ M]k cat (s -1 )k cat /K m cHMBDP+ADPAtIPT4 (WT)8±0,61,58±0,12,1x10 5 cHMBDP+ADPAtIPT4 mut8117±222,44±0,520,7x10 3 cHMBDP+ADPAtIPT4 mut92±0,71,12±0,16,04x10 5 cHMBDP+ATPAtIPT4 mut91,88±0,910,8±0,260x10 6

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 and MSM 6198959216