Association between pituitary adenylate cyclase-activating ...
Characterization of A. thaliana adenylate - isopentenyltransferases
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Transcript of Characterization of A. thaliana adenylate - isopentenyltransferases
Markéta Gemrotová
Isopentenyltransferases (IPT)? Biosynthesis of isoprenoid-type cytokininsTwo types of IPT enzymes:adenylate-isopentenyltransferase(AtIPT1,3,4,5,7,8):
tRNA-isopentenyltransferase: tRNA modification (AtIPT2,AtIPT9)
Localization: AtIPT1,5,8 plastidsAtIPT7 mitochondriaAtIPT2,9 a 4 v cytosolAtIPT3 farnesylated in nucleus/cytoplasmAtIPT3 plastids
Gene s for IPT found in bacteria (A.tumefaciens, R.fascians…), yeasts S. cerevisisiae, tRNA-IPT even in human DNA.
NOO
OH
O-
O-
O
P N
NN
NH2
OH
NOO
OH
O-
O-
O
P N
NN
NH
OH
OP
O
O
O-
PO O
-
O-
+2-3
+ P
O-
O
OHOPO
O-
O-
2-3
DMAPP ATP/ADP iPRTP/iPRDP PPi
A t aden ine-IPT s
Mg 2+
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, stability and weak expression discouraged deeper exploration
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 :
OPOO
-
O
PO
O-
O-
OPOO
-
O
PO
O-
O-
OH
OP
OO
-
O
PO
O-
O-
OH (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
Purification of AtIPT1, AtIPT4, AtIPT5, AtIPT8
IPT1
IPT4
IPT5 IPT8
AtIPT1
AtIPT8
AtIPT4
AtIPT5
AMP ADP ATP
AMP ADP ATP
AtIPT1 AtIPT4
AtIPT5 AtIPT8
Site-directed mutagenesisAtIPT1 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
AtIPT4
cHMBDP +ATPin Ser mutant
cHMBDP +ATPin Gly mutant
Substrates Enzyme Km[µM] kcat(s-1) kcat/Km
cHMBDP+ADP AtIPT4 (WT) 8±0,6 1,58±0,1 2,1x105
cHMBDP+ADP AtIPT4 mut8 117±22 2,44±0,5 20,7x103
cHMBDP+ADP AtIPT4 mut9 2±0,7 1,12±0,1 6,04x105
cHMBDP+ATP AtIPT4 mut9 1,88±0,9 10,8±0,2 60x106
Light at the end of the tunnel ?Characterization of AtIPT3
Expression of IPT3 in S. cerevisiae to get farnesylated protein.
In progress
Acknowledgements:Laboratory of Growth Regulators, Palacký University & Institute of
Experimental Botany AS CR :Lukáš SpíchalVáclav MikMiroslav Strnad
Leibniz-Institute of Plant Biochemistry, Department of Bio-Organic Chemistry, Halle:
Dimitar Vasilev Robert KleinLudger 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