Vacuolar and cytosolic cytokinin dehydrogenases of Arabidopsis thaliana: heterologous expression, purification and properties
Language English Country Great Britain, England Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
20825956
DOI
10.1016/j.phytochem.2010.08.013
PII: S0031-9422(10)00313-4
Knihovny.cz E-resources
- MeSH
- Arabidopsis enzymology genetics metabolism MeSH
- Cytokinins metabolism MeSH
- Electrophoresis, Capillary MeSH
- Escherichia coli enzymology genetics MeSH
- Plants, Genetically Modified enzymology genetics MeSH
- Oxidoreductases genetics metabolism MeSH
- Pichia enzymology genetics MeSH
- Recombinant Proteins metabolism MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
- Nicotiana enzymology genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- cytokinin oxidase MeSH Browser
- Cytokinins MeSH
- Oxidoreductases MeSH
- Recombinant Proteins MeSH
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.
References provided by Crossref.org
IPT9, a cis-zeatin cytokinin biosynthesis gene, promotes root growth
Cytokinin N-glucosides: Occurrence, Metabolism and Biological Activities in Plants
New Insights Into the Metabolism and Role of Cytokinin N-Glucosides in Plants
Auxins and Cytokinins-The Role of Subcellular Organization on Homeostasis
