Laboratoř růstových regulátorů, PřF UP Olomouc a ÚEB AV ČR

Laboratory of Growth Regulators, Faculty of Science, Palacký University in Olomouc and Institute of Experimental Botany ASCR, Czech Republic

Xanthine dehydrogenase and cytokinin metabolism

Jitka Frébortová

Cytokinin meeting, Olomouc, September 8, 2011

Xanthine oxidoreductase

NHN

NNH

O

NHN

NNH

O

O

Hypoxanthine

NHN

NNH

O

O

O

Xanthine Uric acid

XO/XDH XO/XDH

Werner and Witte (2011) Trends Plant Sci. 16, 381-387.

Interaction of cytokinins with bovine milk xanthine oxidase

NHN

NN

NHR

NHN

NN

NHR

OHNHN

NN

NHR

OH

OH

max = 269 nm max = 275 nm max = 306 nm(pH 7, iP, tZ)

Chen et al. (1975) Biochemistry 14, 3088-3093.

Cytokinins described also as inhibitors of bovine milk XO Conversion of cytokinins to 8-hydroxyderivatives confirmed

also in rhizobacterium Serratia protemaculans

XO XO

Sheu et al. (1996) Anticancer Res. 16, 3571-3576. Sheu et al. (1997) Anticancer Res. 17, 1043-1050.Taylor et al. (2006) Phytochemistry 67, 1887-1894.

Plant xanthine dehydrogenase and cytokinin metabolism

Is AtXDH1 converting cytokinins?

Are cytokinins substrates or inhibitors of XORs?

Are the relevant metabolites present in plants?

Biological activity of 8-OH and 2,8-OH cytokinins

Cytokinin free bases are substrates of AtXDH1 and milk XO

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

AtXDH1 Milk XO

Substrate Km (M)

Vmax (pkat/mg)

Km (M)

Vmax (pkat/mg)

Isopentenyladenine 118.1 61.3 34.4 241.8

8-hydroxyisopentenyladenine 62.5 76.9 13.9 124.5

t-Zeatin 95.0 51.7 64.3 396.1

8-hydroxy t-zeatin 176.4 105.3 25.9 282.4

c-Zeatin 146.3 43.7 43.6 211.8

8-hydroxy c-zeatin 59.4 50.5 7.9 52.9

Dihydrozeatin 225.0 97.1 184.0 262.7

Kinetin 76.2 24.4 59.5 78.4

8-hydroxykinetin 54.7 25.6 8.9 12.9

Benzyladenine - - 247.0 255.3

8-hydroxybenzyladenine 28.8 17.3 32.4 21.5

Xanthine 21.7 43500.0 7.5 3844.7

Kinetic constants for the oxidation of cytokinins and xanthine by AtXDH1 and milk XO

Chromatographic analysis of t-zeatin oxidation by AtXDH1

275 nm

310 nm

N9-ribosides, ribotides and glucosides are not oxidized

N9-substituted cytokinins do not interact with AtXDH1 and milk XO

AtXDH1 Milk XO

Cytokinin (inhibitor) Activity (nkat/mg)

Remainining activity (%)

Remaining activity (%)a

None 20.92 ± 0.40 100.0 100

Isopentenyladenine-9-riboside 20.59 ± 0.43 98.4 100

Isopentenyladenine-9-riboside- 5´-monophosphate

21.13 ± 0.52 101.0 -

Isopentenyladenine-9-glucoside 22.13 ± 1.98 105.8 -

t-Zeatin-9-riboside 22.03 ± 1.92 105.3 95

Benzyladenine-9-riboside 20.32 ± 0.74 97.1 100

Kinetin-9-riboside 21.38 ± 0.44 102.2 -

o-Topolin-9-riboside 22.88 ± 0.01 109.4 35

t-Zeatin 11.16 ± 1.45 53.3 75

Effect of N9–substituted cytokinins on xanthine oxidation by AtXDH1 and milk XO

a Data taken from Sauer et al. (2002) Plant Physiol. Biochem. 40, 393-400.

Cytokinin IC50 (M) Isopentenyladenine 5.4

t-Zeatin > 200

c-Zeatin 55.3

Dihydrozeatin >> 200

Benzyladenine >> 200

Kinetin 110

8-hydroxyisopentenyladenine 3.4

8-hydroxy-t-zeatin 40.0

8-hydroxy-c-zeatin 19.2

8-hydroxybenzyladenine 17.4

8-hydroxykinetin 8.9

Topolins > 200

Cytokinins may interfere with normal purine metabolism

Inhibition of uric acid formation by AtXDH1 using cytokinins as inhibitors

Plant xanthine dehydrogenase and cytokinin metabolism

Is AtXDH1 converting cytokinins?

Are cytokinins substrates or inhibitors of XORs?

Are the relevant metabolites present in plants? Is there any physiological function for interaction of

cytokinins with XDH?

Biological activity of 8-OH and 2,8-OH cytokinins

Acknowledgements

Laboratory of Growth Regulators

Lukáš Spíchal

Palacký University/ Institute of Experimental Botany

Technische Universität Braunschweig

Florian Bittner

Palacký University

Department of Biochemistry

Petr TarkowskiPavel Jaworek

Institut für Pflanzenbiologie