caa a22 4500 463160282 CHVBK 20180406164745.0 cr unu---uuuuu 170326e20071201xx s 000 0 eng 10.1007/s10535-007-0141-6 doi (NATIONALLICENCE)springer-10.1007/s10535-007-0141-6 Functional expression and purification of cytokinin dehydrogenase from Arabidopsis thaliana (AtCKX2) in Saccharomyces cerevisiae [Elektronische Daten] [J. Frébortová, P. Galuszka, T. Werner, T. Schmülling, I. Frébort] Cytokinin dehydrogenase (CKX) is responsible for regulating the endogenous cytokinin content by oxidative removal of the side chain and seven distinct genes, AtCKX1 to AtCKX7, code for the enzyme in Arabidopsis thaliana. The recombinant enzyme AtCKX2 was produced in Saccharomyces cerevisiae after expressing the corresponding gene from a plasmid (pDR197) or following chromosomal integration, under either the constitutive promoter PMA1 or the inducible promoter GAL1. The recombinant protein was purified from yeast culture media using a sequence of chromatographic steps. The purified enzyme had a molecular mass of 61 kDa and a typical flavoprotein spectrum. The specific activity of the enzyme was 87.8 µkat g−1, with isopentenyladenine as a substrate and 2,3-dimethoxy-5-methyl-p-benzoquinone as an electron acceptor. The pH optimum lay between 7.0 and 8.0, depending on the electron acceptor used. AtCKX2 reacts both with isoprenoid and aromatic cytokinins, the activity with isoprenoid cytokinins being two to three orders of magnitude higher. AtCKX2 prefers p-quinones and the synthetic dye 2,6-dichlorophenol indophenol as electron acceptors, although low reactivity with oxygen can also be observed. This study presents the first purification and characterization of the enzyme from Arabidopsis thaliana. Institute of Experimental Botany, ASCR, 2007 cytokinin metabolism nationallicence cytokinin oxidase nationallicence AtCKX2 : cytokinin dehydrogenase from Arabidopsis thaliana encoded by the gene AtCKX2 (TAIR accession No. At2g19500) nationallicence CKX : cytokinin dehydrogenase (EC 1.5.99.12) nationallicence DCPIP : 2,6-dichlorophenol indophenol nationallicence DHZ : dihydrozeatin nationallicence iP : N 6-(2-isopentenyl)adenine nationallicence iP9G : N 6-(2-isopentenyl)adenine 9-glucoside nationallicence iPR : N 6-(2-isopentenyl)adenine 9-riboside nationallicence iPRMP : N 6-(2-isopentenyl)adenine 9-riboside-5′-monophosphate nationallicence MiP : N 6-methyl-isopentenyladenine nationallicence m -T : m-topolin nationallicence NBT : nitroblue tetrazolium chloride nationallicence o -T : o-topolin nationallicence p -T : p-topolin nationallicence Q0 : 2,3-dimethoxy-5-methyl-1,4-benzoquinone nationallicence t -Z : trans-zeatin nationallicence t -ZR : trans-zeatin 9-riboside nationallicence Z9G : trans-zeatin 9-glucoside nationallicence ZmCKX1 : cytokinin dehydrogenase from Zea mays encoded by the gene ZmCKX1 (GenBank accession no. AF044603) nationallicence Frébortová J. Laboratory of Growth Regulators, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-78371, Olomouc, Czech Republic aut Galuszka P. Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-78371, Olomouc, Czech Republic aut Werner T. Institute of Biology and Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, D-14195, Berlin, Germany aut Schmülling T. Institute of Biology and Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, D-14195, Berlin, Germany aut Frébort I. Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-78371, Olomouc, Czech Republic aut Biologia Plantarum Springer Netherlands 51/4(2007-12-01), 673-682 0006-3134 51:4<673 2007 51 10535 https://doi.org/10.1007/s10535-007-0141-6 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10535-007-0141-6 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Frébortová J. Laboratory of Growth Regulators, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-78371, Olomouc, Czech Republic aut NATIONALLICENCE 700 1- Galuszka P. Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-78371, Olomouc, Czech Republic aut NATIONALLICENCE 700 1- Werner T. Institute of Biology and Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, D-14195, Berlin, Germany aut NATIONALLICENCE 700 1- Schmülling T. Institute of Biology and Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, D-14195, Berlin, Germany aut NATIONALLICENCE 700 1- Frébort I. Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-78371, Olomouc, Czech Republic aut NATIONALLICENCE 773 0- Biologia Plantarum Springer Netherlands 51/4(2007-12-01), 673-682 0006-3134 51:4<673 2007 51 10535 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer