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. 2004 May 15;380(Pt 1):121–130. doi: 10.1042/BJ20031813

Catalytic reaction of cytokinin dehydrogenase: preference for quinones as electron acceptors.

Jitka Frébortová 1, Marco W Fraaije 1, Petr Galuszka 1, Marek Sebela 1, Pavel Pec 1, Jan Hrbác 1, Ondrej Novák 1, Kristin D Bilyeu 1, James T English 1, Ivo Frébort 1
PMCID: PMC1224151  PMID: 14965342

Abstract

The catalytic reaction of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) 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 2,6-dichlorophenol indophenol, 2,3-dimethoxy-5-methyl-1,4-benzoquinone or 1,4-naphthoquinone as electron acceptor, turnover rates with N6-(2-isopentenyl)adenine of approx. 150 s(-1) could be obtained. This suggests that the natural electron 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 (k(red)=950 s(-1)). Re-oxidation of the reduced enzyme by molecular oxygen is too slow to be of physiological relevance, confirming its 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 catalytic mode for 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.

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Selected References

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  1. Aikazyan V. T., Nalbandyan R. M. Plantacyanin from spinach. FEBS Lett. 1975 Jul 15;55(1):272–274. doi: 10.1016/0014-5793(75)81009-x. [DOI] [PubMed] [Google Scholar]
  2. Benson T. E., Filman D. J., Walsh C. T., Hogle J. M. An enzyme-substrate complex involved in bacterial cell wall biosynthesis. Nat Struct Biol. 1995 Aug;2(8):644–653. doi: 10.1038/nsb0895-644. [DOI] [PubMed] [Google Scholar]
  3. Bilyeu K. D., Cole J. L., Laskey J. G., Riekhof W. R., Esparza T. J., Kramer M. D., Morris R. O. Molecular and biochemical characterization of a cytokinin oxidase from maize. Plant Physiol. 2001 Jan;125(1):378–386. doi: 10.1104/pp.125.1.378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Binda Claudia, Mattevi Andrea, Edmondson Dale E. Structure-function relationships in flavoenzyme-dependent amine oxidations: a comparison of polyamine oxidase and monoamine oxidase. J Biol Chem. 2002 May 15;277(27):23973–23976. doi: 10.1074/jbc.R200005200. [DOI] [PubMed] [Google Scholar]
  5. Coulombe R., Yue K. Q., Ghisla S., Vrielink A. Oxygen access to the active site of cholesterol oxidase through a narrow channel is gated by an Arg-Glu pair. J Biol Chem. 2001 Jun 7;276(32):30435–30441. doi: 10.1074/jbc.M104103200. [DOI] [PubMed] [Google Scholar]
  6. Doerge D. R., Divi R. L., Churchwell M. I. Identification of the colored guaiacol oxidation product produced by peroxidases. Anal Biochem. 1997 Jul 15;250(1):10–17. doi: 10.1006/abio.1997.2191. [DOI] [PubMed] [Google Scholar]
  7. Dwyer T. M., Mortl S., Kemter K., Bacher A., Fauq A., Frerman F. E. The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer. Biochemistry. 1999 Jul 27;38(30):9735–9745. doi: 10.1021/bi9903906. [DOI] [PubMed] [Google Scholar]
  8. Dym O., Pratt E. A., Ho C., Eisenberg D. The crystal structure of D-lactate dehydrogenase, a peripheral membrane respiratory enzyme. Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9413–9418. doi: 10.1073/pnas.97.17.9413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eckardt Nancy A. ATM to the rescue: repairing DNA damage. Plant Cell. 2003 Jan;15(1):1–3. doi: 10.1105/tpc.150110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fraaije M. W., Mattevi A. Flavoenzymes: diverse catalysts with recurrent features. Trends Biochem Sci. 2000 Mar;25(3):126–132. doi: 10.1016/s0968-0004(99)01533-9. [DOI] [PubMed] [Google Scholar]
  11. Fraaije M. W., Van Berkel W. J., Benen J. A., Visser J., Mattevi A. A novel oxidoreductase family sharing a conserved FAD-binding domain. Trends Biochem Sci. 1998 Jun;23(6):206–207. doi: 10.1016/s0968-0004(98)01210-9. [DOI] [PubMed] [Google Scholar]
  12. Fraaije M. W., van Berkel W. J. Catalytic mechanism of the oxidative demethylation of 4-(methoxymethyl)phenol by vanillyl-alcohol oxidase. Evidence for formation of a p-quinone methide intermediate. J Biol Chem. 1997 Jul 18;272(29):18111–18116. doi: 10.1074/jbc.272.29.18111. [DOI] [PubMed] [Google Scholar]
  13. Fraaije M. W., van den Heuvel R. H., van Berkel W. J., Mattevi A. Covalent flavinylation is essential for efficient redox catalysis in vanillyl-alcohol oxidase. J Biol Chem. 1999 Dec 10;274(50):35514–35520. doi: 10.1074/jbc.274.50.35514. [DOI] [PubMed] [Google Scholar]
  14. Frébort Ivo, Sebela Marek, Galuszka Petr, Werner Tomás, Schmülling Thomas, Pec Pavel. Cytokinin oxidase/cytokinin dehydrogenase assay: optimized procedures and applications. Anal Biochem. 2002 Jul 1;306(1):1–7. doi: 10.1006/abio.2002.5670. [DOI] [PubMed] [Google Scholar]
  15. Galuszka P., Frébort I., Sebela M., Sauer P., Jacobsen S., Pec P. Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals. Eur J Biochem. 2001 Jan;268(2):450–461. doi: 10.1046/j.1432-1033.2001.01910.x. [DOI] [PubMed] [Google Scholar]
  16. Gutierrez A., Lian L. Y., Wolf C. R., Scrutton N. S., Roberts G. C. Stopped-flow kinetic studies of flavin reduction in human cytochrome P450 reductase and its component domains. Biochemistry. 2001 Feb 20;40(7):1964–1975. doi: 10.1021/bi001719m. [DOI] [PubMed] [Google Scholar]
  17. Houba-Hérin N., Pethe C., d'Alayer J., Laloue M. Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts. Plant J. 1999 Mar;17(6):615–626. doi: 10.1046/j.1365-313x.1999.00408.x. [DOI] [PubMed] [Google Scholar]
  18. Lancaster C. R., Michel H. Refined crystal structures of reaction centres from Rhodopseudomonas viridis in complexes with the herbicide atrazine and two chiral atrazine derivatives also lead to a new model of the bound carotenoid. J Mol Biol. 1999 Feb 26;286(3):883–898. doi: 10.1006/jmbi.1998.2532. [DOI] [PubMed] [Google Scholar]
  19. Marchler-Bauer Aron, Panchenko Anna R., Shoemaker Benjamin A., Thiessen Paul A., Geer Lewis Y., Bryant Stephen H. CDD: a database of conserved domain alignments with links to domain three-dimensional structure. Nucleic Acids Res. 2002 Jan 1;30(1):281–283. doi: 10.1093/nar/30.1.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Massey V., Palmer G. On the existence of spectrally distinct classes of flavoprotein semiquinones. A new method for the quantitative production of flavoprotein semiquinones. Biochemistry. 1966 Oct;5(10):3181–3189. doi: 10.1021/bi00874a016. [DOI] [PubMed] [Google Scholar]
  21. Mok David WS, Mok Machteld C. CYTOKININ METABOLISM AND ACTION. Annu Rev Plant Physiol Plant Mol Biol. 2001 Jun;52(NaN):89–118. doi: 10.1146/annurev.arplant.52.1.89. [DOI] [PubMed] [Google Scholar]
  22. Morris R. O., Bilyeu K. D., Laskey J. G., Cheikh N. N. Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochem Biophys Res Commun. 1999 Feb 16;255(2):328–333. doi: 10.1006/bbrc.1999.0199. [DOI] [PubMed] [Google Scholar]
  23. Motyka V., Faiss M., Strand M., Kaminek M., Schmulling T. Changes in Cytokinin Content and Cytokinin Oxidase Activity in Response to Derepression of ipt Gene Transcription in Transgenic Tobacco Calli and Plants. Plant Physiol. 1996 Nov;112(3):1035–1043. doi: 10.1104/pp.112.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pace C. P., Stankovich M. T. Redox properties of electron-transferring flavoprotein from Megasphaera elsdenii. Biochim Biophys Acta. 1987 Feb 25;911(3):267–276. doi: 10.1016/0167-4838(87)90067-7. [DOI] [PubMed] [Google Scholar]
  25. Schmülling Thomas. New Insights into the Functions of Cytokinins in Plant Development. J Plant Growth Regul. 2002 Mar;21(1):40–49. doi: 10.1007/s003440010046. [DOI] [PubMed] [Google Scholar]
  26. Werner T., Motyka V., Strnad M., Schmülling T. Regulation of plant growth by cytokinin. Proc Natl Acad Sci U S A. 2001 Aug 14;98(18):10487–10492. doi: 10.1073/pnas.171304098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yang Shu Hua, Yu Hao, Goh Chong Jin. Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid. Plant Mol Biol. 2003 Jan;51(2):237–248. doi: 10.1023/a:1021115816540. [DOI] [PubMed] [Google Scholar]
  28. Yang Shu Hua, Yu Hao, Goh Chong Jin. Isolation and characterization of the orchid cytokinin oxidase DSCKX1 promoter. J Exp Bot. 2002 Sep;53(376):1899–1907. doi: 10.1093/jxb/erf055. [DOI] [PubMed] [Google Scholar]
  29. Yin Y., Sampson N. S., Vrielink A., Lario P. I. The presence of a hydrogen bond between asparagine 485 and the pi system of FAD modulates the redox potential in the reaction catalyzed by cholesterol oxidase. Biochemistry. 2001 Nov 20;40(46):13779–13787. doi: 10.1021/bi010843i. [DOI] [PubMed] [Google Scholar]

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