|Frebortova, Jitka - PALACKY UNIV-CZECH REPUB|
|Fraaije, Marco - U OF GRONINGEN-NETHERLAND|
|Galuszka, Petr - PALACKY UNIV-CZECH REPUB|
|Sebela, Marek - PALACKY UNIV-CZECH REPUB|
|Pec, Pavel - PALACKY UNIV-CZECH REPUB|
|Hrbac, Jan - PALACKY UNIV-CZECH REPUB|
|Novak, Ondrej - PALACKY UNIV-CZECH REPUB|
|English, James - UNIV OF MISSOURI-COLUMBIA|
|Frebort, Ivo - PALACKY UNIV-CZECH REPUB|
Submitted to: Biochemical Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 13, 2004
Publication Date: May 15, 2004
Citation: Frebortova, J., Fraaije, M., Galuszka, P., Sebela, M., Pec, P., Hrbac, J., Novak, O., Bilyeu, K.D., English, J., Frebort, I. 2004. Catalytic reaction of cytokinin dehydrogenase: preference for quinones as electron acceptors. Biochemical Journal. 380(1):121-130. Interpretive Summary: A multitude of enzyme-catalyzed biochemical reactions are carried out in every compartment of plant cells. In their active state, the cytokinin plant hormones provide a signal for cell division. Because of the presence of extremely low levels of cytokinins in plant tissues, a comprehensive understanding of their role in plant development has been somewhat limited. Cytokinins are irreversibly inactivated through the activity of cytokinin oxidase/dehydrogenase enzymes. The enzymes have been historically classified as oxidases, but recent work has demonstrated that oxygen is a poor participant in the reactions. The objective of this work was to dissect the cytokinin oxidase/dehydrogenase enzymatic reaction mechanism to gain an understanding of the basic biochemistry involved in regulating cytokinin levels and thus, plant cell division. The enzyme was studied for its electrochemical properties in connection with spectral changes associated with its flavin cofactor. When the reaction rate was examined for each half-reaction, the experiments revealed that the enzyme initially acts very efficiently on its substrate and then is limited by reoxidation by oxygen or an alternative electron acceptor. The results indicate that efficient inactivation of cytokinins by cytokinin oxidase/dehydrogenase may secondarily depend on the localized presence of a suitable electron acceptor. The results will benefit researchers with the ability to manipulate the cell divisions associated with seed development, and therefore, possibly increase seed quality traits and yield.
Technical Abstract: The catalytic reaction of cytokinin oxidase/dehydrogenase (EC 188.8.131.52) was studied in detail using the recombinant flavoenzyme from maize. Determination of the redox potential of the covalently linked flavin cofactor revealed a relatively high potential dictating the type of electron acceptor that can be used by the enzyme. Using DCPIP; 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q0); or 1,4-naphtoquinone as electron acceptor, turnover rates with N6-(2-isopentenyl)adenine (iP) of around 150 s-1 could be obtained. This suggests that the natural electron acceptor of the enzyme is most likely a p-quinone or similar compound. By using the stopped-flow technique it was found that the enzyme is rapidly reduced by N6-(2-isopentenyl)adenine (kred = 950 s-1). Reoxidation of the reduced enzyme by molecular oxygen is too slow to be of physiological relevance, confirming the classification as a dehydrogenase. Furthermore, it was established for the first time that the enzyme is capable of degrading aromatic cytokinins, although at low reaction rates. As a result the enzyme displays a dual manner of oxidative degradation of cytokinins: a low-rate and low-substrate specificity reaction with oxygen as the electron acceptor, and high activity and strict specificity for isopentenyladenine and analogous cytokinins with some specific electron acceptors.