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Catalytic reaction of cytokinin dehydrogenase Frébortová, Jitka; Fraaije, Marco W.; Galuszka, Petr; Šebela, Marek; Peč, Pavel; Hrbáč, Jan; Novák, Ondřej; Bilyeu, Kristin D.; English, James T.; Frébort, Ivo Published in: Biochemical Journal
DOI: 10.1042/BJ20031813
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Publication date: 2004
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Citation for published version (APA): Frébortová, J., Fraaije, M. W., Galuszka, P., Šebela, M., Peč, P., Hrbáč, J., ... Frebort, N. V. (2004). Catalytic reaction of cytokinin dehydrogenase: preference for quinones as electron acceptors. Biochemical Journal, 380, 121 - 130. https://doi.org/10.1042/BJ20031813
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Download date: 12-11-2019 Biochem. J. (2004) 380, 121–130 (Printed in Great Britain) 121
Catalytic reaction of cytokinin dehydrogenase: preference for quinones as electron acceptors
Jitka FREBORTOV´ A*,´ Marco W. FRAAIJE†, Petr GALUSZKA‡, Marek SEBELAˇ §, Pavel PECˇ§, Jan HRBA´Cˇ ,Ondrejˇ NOVAK*,´ Kristin D. BILYEU¶, James T. ENGLISH** and Ivo FREBORT´ ‡1 *Laboratory of Growth Regulators, Faculty of Science, Palacky´ University/Institute of Experimental Botany of the Academy of Science, Slechtitelˇ u˚ 11, 783 71 Olomouc, Czech Republic, †Laboratory of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands, ‡Division of Molecular Biology, Faculty of Science, Palacky´ University, Slechtitelˇ u˚ 11, 783 71 Olomouc, Czech Republic, §Department of Biochemistry, Faculty of Science, Palacky´ University, Slechtitelˇ u˚ 11, 783 71 Olomouc, Czech Republic, Department of Physical Chemistry, Faculty of Science, Palacky´ University, tˇr. Svobody 26, 771 46 Olomouc, Czech Republic, ¶USDA/ARS, 210 Waters Hall, Columbia, MO 65211, U.S.A., and **Department of Plant Microbiology and Pathology, University of Missouri, Columbia, MO 65211, U.S.A.
The catalytic reaction of cytokinin oxidase/dehydrogenase (EC oxygen is too slow to be of physiological relevance, confirming its 1.5.99.12) was studied in detail using the recombinant flavo- classification as a dehydrogenase. Furthermore, it was established enzyme from maize. Determination of the redox potential of the for the first time that the enzyme is capable of degrading aromatic covalently linked flavin cofactor revealed a relatively high poten- cytokinins, although at low reaction rates. As a result, the enzyme tial dictating the type of electron acceptor that can be used by the displays a dual catalytic mode for oxidative degradation of cyto- enzyme. Using 2,6-dichlorophenol indophenol, 2,3-dimethoxy-5- kinins: a low-rate and low-substrate specificity reaction with methyl-1,4-benzoquinone or 1,4-naphthoquinone as electron ac- oxygen as the electron acceptor, and high activity and strict ceptor, turnover rates with N6-(2-isopentenyl)adenine of approx. specificity for isopentenyladenine and analogous cytokinins with 150 s−1 could be obtained. This suggests that the natural electron some specific electron acceptors. acceptor of the enzyme is quite probably 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 Key words: cytokinin dehydrogenase (cytokinin oxidase), cyto- −1 (kred = 950 s ). Re-oxidation of the reduced enzyme by molecular kinin, flavoprotein, plant hormone metabolism, quinone.
INTRODUCTION atically in Figure 1 [4]. CKX was revealed to be a member of the oxidoreductase family after the enzyme was cloned from maize Both prokaryotes and eukaryotes have been shown to utilize and shown to contain the FAD-binding domain and a covalent flavoprotein enzymes for a wide variety of oxidoreductase association with flavin [5,9]. To avoid confusion with the use of reactions. A family of flavin-containing oxidoreductases was pre- the traditional and the new names, only the abbreviation CKX viously identified on the basis of the presence of a shared FAD- will be used throughout the paper. binding domain [1]. Members of this group include enzymes with CKX removes the isoprenoid side chain of iP [N6-(2-isopen- covalently and non-covalently bound FAD capable of performing tenyl)adenine] and zeatin, converting them into adenine and the oxidation as well as dehydrogenation reactions, but it is not corresponding aldehyde [3,7,8]. Genes encoding the enzyme were clear how the FAD-binding domain provides access to the elec- cloned from maize [9,10], A. thaliana [5,11,12], Dendrobium tron acceptor. Electron transport resulting in re-oxidation of FAD orchid [13,14] and recently also from barley (P. Galuszka, appears to be a divergent feature of this group of enzymes. To unpublished work; GenBank® accession no. AF491590). To date, understand the evolution of different mechanisms of enzyme CKX was not known to act on aromatic side-chain cytokinins flavin utilization, it is important to compare the reaction mech- such as kinetin and benzyladenine derivatives [7]. anisms and physical properties of different enzymes containing Recently, it has been shown that the addition of DCPIP (2,6- the FAD-binding domain. dichlorophenol indophenol) to in vitro reaction increases the re- Cytokinins are important plant hormones [2], which are select- action rate of degradation of trans-zeatin by the ZmCKX1 [the ively inactivated by oxidative cleavage of their side chain. The CKX enzyme from maize, encoded by the gene ZmCXK1 (Gen- enzyme catalysing this degradation has been historically classified Bank® accession no. AF044603)] nearly 4000-fold faster than as a cytokinin oxidase [3], but recent findings indicate that the the oxygen-dependent rate [6]. In the present study, we present enzyme uses an electron acceptor other than oxygen [4–6]; re- further characterizations of the reaction mechanism of CXK from classification of the enzyme as cytokinin dehydrogenase (CKX; maize, ZmCKX1, which focus on explaining the kinetic and EC 1.5.99.12) was approved by the Nomenclature Committee of redox properties of the enzyme and the role of the electron accep- the International Union of Biochemistry and Molecular Biology tor. These studies form the basis for broader comparisons among in 2001. The reaction catalysed by the enzyme is shown schem- FAD oxidoreductases and within the plant CKX gene family.
Abbreviations used: CAN, citraconic anhydride; CKX, cytokinin dehydrogenase (EC 1.5.99.12); CPPU, N-(2-chloro-4-pyridyl)-N9-phenylurea; DCPIP, 2,6-dichlorophenol indophenol; DEPC, diethyl pyrocarbonate; EDC, N-(3-dimethyl-aminopropyl)-N-ethylcarbodi-imide; iP, N6-(2-isopentenyl)adenine; iPR, N6-(2-isopentenyl)adenosine; NAI, N-acetylimidazole; NBS, N-bromosuccinimide; NHE, normal hydrogen electrode; NEM, N-ethylmaleimide; PHG, phenylglyoxal; Q0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone; Q1, 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone; SCE, saturated calomel electrode. 1 To whom correspondence should be addressed (e-mail [email protected]).