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