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University of Groningen Catalytic Reaction of Cytokinin View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Groningen University of Groningen 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 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2004 Link to publication in University of Groningen/UMCG research database 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 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. 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]). c 2004 Biochemical Society 122 J. Frebortov´ a´ and others Figure 1 Reaction scheme of CKX (EC 1.5.99.12) EXPERIMENTAL 2-methyl-1,4-naphthoquinone), coenzyme Q0 (2,3-dimethoxy- 5-methyl-1,4-benzoquinone), NAI (N-acetylimidazole), NEM Enzymes and chemicals (N-ethylmaleimide), kaempferol [3,5,7-trihydroxy-2-(4-hydroxy- The enzyme, recombinant ZmCKX1 produced by Pichia pastoris, phenyl)-4H-1-benzopyran-4-one], 4-methylcatechol, Diuron [3- was purified as described previously [5] and stored as a concen- (3,4-dichlorophenyl)-1,1-dimethylurea], guaiacol (2-methoxy- trated stock solution (87.7 µM) in TE buffer (10 mM Tris/HCl, phenol), tyrosine, xanthine, Methyl Viologen, thionin, Methylene 1 mM EDTA, pH 8.0), containing 1 M KCl at 4 ◦C. Partially Blue, resorufin and other chemicals were from Sigma. purified plantacyanin from spinach leaves was prepared by the method described in [15]. Horse heart cytochrome c was obtained Differential pulse polarography from Biomed (Cracow, Poland); bovine milk xanthine oxidase (EC 1.1.3.22) was from Sigma (St. Louis, MO, U.S.A.). Differential pulse polarography measurements were performed on Rosmaric acid, scopoletin, caffeic acid, daphnoretin, o- and ZmCKX1 in a PRG 4 apparatus (Tacussel, Villeurbane, France) p-coumaric acid, luteolin, hydrocaffeic acid and umbelliferone as described previously [17], using a mercury dropping electrode were donated by Dr Lubom´ır Opletal (Charles University, Hradec and SCE (saturated calomel electrode) as the reference. The reaction mixture in a glass polarographic cell (2 ml) contained Kralov´ e,´ Czech Republic). Coenzyme Q1 [2,3-dimethoxy-5- methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone] was a gift ZmCKX1 (1.5 µM) in 43 mM Mops/NaOH (pH 7.2) and from Professor Kazunobu Matsushita (Yamaguchi University, 0.24 mM EDTA. The reaction mixture was de-aerated by bubbling Japan) and atrazine was donated by Dr Pavel Adamovsky´ (Depart- with argon gas for 10 min and polarographed over the potential ment of Analytical Chemistry, Palacky´ University, Olomouc, range between − 1.4 and 0.0 V. Czech Republic). 1,4-Naphthoquinone was obtained from BDH Chemicals (Poole, Dorset, U.K.) and DCPIP and indole-3-acetic acid were from LOBA Feinchemie (Fischamend, Austria).
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