Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals.
An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purified wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize. N6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested. Interestingly, oxygen was not required and hydrogen peroxide not produced during the catalytic reaction, so the enzyme behaves as a dehydrogenase rather than an oxidase. This was confirmed by the ability of the enzyme to transfer electrons to artificial electron acceptors, such as phenazine methosulfate and 2,6-dichlorophenol-indophenol. 2,3-Dimethoxy-5-methyl-1,4-benzoquinone, a precursor of the naturally occurring electron acceptor ubiquinone, readily interacts with the enzyme in micromolar concentrations. Typical flavoenzyme inhibitors such as acriflavine and diphenyleneiodonium inhibited this enzyme activity. Presence of the flavin cofactor in the enzyme was confirmed by differential pulse polarography and by measuring the fluorescence emission spectrum. Possible existence of a second redox centre is discussed.
Galuszka
P
P
Department of Biochemistry, Faculty of Science, Palacký University, Slechtitelu Olomouc, Czech Republic.
Frébort
I
I
Sebela
M
M
Sauer
P
P
Jacobsen
S
S
Pec
P
P
D000595:Amino Acid Sequence; D003583:Cytokinins / Q000378:metabolism*; D002523:Edible Grain / Q000201:enzymology*; D004579:Electron Transport; D005420:Flavoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D017343:Genes, Plant; D001467:Hordeum / Q000201:enzymology; D006861:Hydrogen Peroxide / Q000032:analysis; D007541:Isopentenyladenosine / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology
D003583:Cytokinins; D005420:Flavoproteins; D007541:Isopentenyladenosine; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; C019984:cytokinin oxidase