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 sta ining 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 thalian a, but not to the enzyme from maize. N-6-isopentenyl-2-(2-hydroxyethylamino )-9-methyladenine is the best substrate from all the cytokinins tested. Int erestingly, oxygen was not required and hydrogen peroxide not produced duri ng the catalytic reaction, so the enzyme behaves as a dehydrogenase rather than an oxidase. This was confirmed by the ability of the enzyme to transfe r electrons to artificial electron acceptors, such as phenazine methosulfat e and 2,6-dichlorophenol-indophenol. 2,3-Dimethoxy-5-methyl-1,4-benzoquinon e, a precursor of the naturally occurring electron acceptor ubiquinone, rea dily interacts with the enzyme in micromolar concentrations. Typical flavoe nzyme inhibitors such as acriflavine and diphenyleneiodonium inhibited this enzyme activity. Presence of the flavin cofactor in the enzyme was confirm ed by differential pulse polarography and by measuring the fluorescence emi ssion spectrum. Possible existence of a second redox centre is discussed.