title: Vacuolar and cytosolic cytokinin dehydrogenases of Arabidopsis thaliana: heterologous expression, purification and properties.
abstract: The catabolism of cytokinins is a vital component of hormonal regulation, contributing to the control of active forms of cytokinins and their cellular distribution. The enzyme catalyzing the irreversible cleavage of N(6)-side chains from cytokinins is a flavoprotein classified as cytokinin dehydrogenase (CKX, EC 1.5.99.12). CKXs also show low cytokinin oxidase activity, but molecular oxygen is a comparatively poor electron acceptor. The CKX gene family of Arabidopsis thaliana comprises seven members. Four code for proteins secreted to the apoplast, the remainder are not secreted. Two are targeted to the vacuoles and one is restricted to the cytosol. This study presents the purification and characterization of each of these non-secreted CKX enzymes and substrate specificities are discussed with respect to their compartmentation. Vacuolar enzymes AtCKX1 and AtCKX3 were produced in Pichia pastoris and cytosolic enzyme AtCKX7 was expressed in Escherichia coli. The recombinant proteins were purified by column chromatography. All enzymes preferred synthetic electron acceptors over oxygen, namely potassium ferricyanide and 2,3-dimetoxy-5-methyl-1,4-benzoquinone (Q(0)). In slightly acidic conditions (pH 5.0), N(6)-(2-isopentenyl)adenine 9-glucoside (iP9G) was the best substrate for AtCKX1 and AtCKX7, whereas AtCKX3 preferentially degraded N(6)-(2-isopentenyl)adenine 9-riboside-5'-monophosphate (iPMP). Moreover, vacuolar AtCKX enzymes in certain conditions degraded N(6)-(2-isopentenyl)adenine di- and triphosphates two to five times more effectively than its monophosphate.
authors.affiliation: Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic. marta.kowalska@email.cz
authors.forename: Marta
authors.initials: M
authors.lastname: Kowalska
authors.forename: Petr
authors.initials: P
authors.lastname: Galuszka
authors.forename: Jitka
authors.initials: J
authors.lastname: Frébortová
authors.forename: Marek
authors.initials: M
authors.lastname: Šebela
authors.forename: Tibor
authors.initials: T
authors.lastname: Béres
authors.forename: Tomáš
authors.initials: T
authors.lastname: Hluska
authors.forename: Mária
authors.initials: M
authors.lastname: Šmehilová
authors.forename: Kristin D
authors.initials: KD
authors.lastname: Bilyeu
authors.forename: Ivo
authors.initials: I
authors.lastname: Frébort
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003583:Cytokinins / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics
chemical_list: D003583:Cytokinins; D011994:Recombinant Proteins; D010088:Oxidoreductases; C019984:cytokinin oxidase