Functional expression and purification of cytokinin dehydrogenase from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (AtCKX2) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> |
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Authors: | J Frébortová P Galuszka T Werner T Schmülling I Frébort |
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Institution: | (1) Laboratory of Growth Regulators, Faculty of Science, Palacky University, Šlechtitelů 11, CZ-78371 Olomouc, Czech Republic;(2) Institute of Experimental Botany of the Academy of Science, Šlechtitelů 11, CZ-78371 Olomouc, Czech Republic;(3) Department of Biochemistry, Faculty of Science, Palacky University, Šlechtitelů 11, CZ-78371 Olomouc, Czech Republic;(4) Institute of Biology and Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, D-14195 Berlin, Germany |
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Abstract: | 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. |
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Keywords: | cytokinin metabolism cytokinin oxidase |
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