Functional expression and purification of cytokinin dehydrogenase from Arabidopsis thaliana (AtCKX2) in Saccharomyces cerevisiae
Frebortova, J.,Univerzita Palackeho, Olomouc (Czech Republic). Laborator Rustovych Regulatoru | Galuszka, P.,Univerzita Palackeho, Olomouc (Czech Republic). Katedra Biochemie | Werner, T.,Free Univ. of Berlin (Germany). Inst. of Biology and Applied Genetics | Schmuelling, T.,Free Univ. of Berlin (Germany). Inst. of Biology and Applied Genetics | Frebort, I.,Univerzita Palackeho, Olomouc (Czech Republic). Katedra Biochemie
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 microkat/g, 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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