Refine
Document Type
- Article (1)
- Doctoral Thesis (1)
Language
- English (2)
Is part of the Bibliography
- yes (2) (remove)
Keywords
- AtDGK gene (1)
- AtDGK genes (1)
- Diacylglycerol (1)
- Diacylglycerol-Kinasen (1)
- Phosphatidsäure (1)
- Signaltransduktionsprozesse (1)
- auxin (1)
- diacylglycerol (1)
- phosphatidic acid (1)
- signaling (1)
Institute
- Institut für Biochemie und Biologie (2) (remove)
Diacylglycerol kinase (DGK) regulates the level of the second messenger diacylglycerol and produces phosphatidic acid (PA), another signaling molecule. The Arabidopsis thaliana genome encodes seven putative diacylglycerol kinase isozymes (named AtDGK1 to -7), structurally falling into three major clusters. So far, enzymatic activity has not been reported for any plant Cluster II DGK. Here, we demonstrate that a representative of this cluster, AtDGK7, is biochemically active when expressed as a recombinant protein in Escherichia coli. AtDGK7, encoded by gene locus At4g30340, contains 374 amino acids with an apparent molecular mass of 41.2 kDa. AtDGK7 harbors an N-terminal catalytic domain, but in contrast to various characterized DGKs (including AtDGK2), it lacks a cysteine-rich domain at its N terminus, and, importantly, its C-terminal DGK accessory domain is incomplete. Recombinant AtDGK7 expressed in E. coli exhibits Michaelis-Menten type kinetics with 1,2-dioleoyl-sn-glycerol as substrate. AtDGK7 activity was affected by pH, detergents, and the DGK inhibitor R59022. We demonstrate that both AtDGK2 and AtDGK7 phosphorylate diacylglycerol molecular species that are typically found in plants, indicating that both enzymes convert physiologically relevant substrates. AtDGK7 is expressed throughout the Arabidopsis plant, but expression is strongest in flowers and young seedlings. Expression of AtDGK2 is transiently induced by wounding. R59022 at similar to 80 mu M inhibits root elongation and lateral root formation and reduces plant growth, indicating that DGKs play an important role in plant development
Biochemical and physiological studies of Arabidopsis thaliana Diacylglycerol Kinase 7 (AtDGK7)
(2006)
A family of diacylglycerol kinases (DGK) phosphorylates the substrate diacylglycerol (DAG) to generate phosphatidic acid (PA) . Both molecules, DAG and PA, are involved in signal transduction pathways. In the model plant Arabidopsis thaliana, seven candidate genes (named AtDGK1 to AtDGK7) code for putative DGK isoforms. Here I report the molecular cloning and characterization of AtDGK7. Biochemical, molecular and physiological experiments of AtDGK7 and their corresponding enzyme are analyzed. Information from Genevestigator says that AtDGK7 gene is expressed in seedlings and adult Arabidopsis plants, especially in flowers. The AtDGK7 gene encodes the smallest functional DGK predicted in higher plants; but also, has an alternative coding sequence containing an extended AtDGK7 open reading frame, confirmed by PCR and submitted to the GenBank database (under the accession number DQ350135). The new cDNA has an extension of 439 nucleotides coding for 118 additional amino acids The former AtDGK7 enzyme has a predicted molecular mass of ~41 kDa and its activity is affected by pH and detergents. The DGK inhibitor R59022 also affects AtDGK7 activity, although at higher concentrations (i.e. IC50 ~380 µM). The AtDGK7 enzyme also shows a Michaelis-Menten type saturation curve for 1,2-DOG. Calculated Km and Vmax were 36 µM 1,2-DOG and 0.18 pmol PA min-1 mg of protein-1, respectively, under the assay conditions. Former protein AtDGK7 are able to phosphorylate different DAG analogs that are typically found in plants. The new deduced AtDGK7 protein harbors the catalytic DGKc and accessory domains DGKa, instead the truncated one as the former AtDGK7 protein (Gomez-Merino et al., 2005).