Arabidopsis AtDGK7, the smallest member of plant diacylglycerol kinases (DGKs), displays unique biochemical features and saturates at low substrate concentration: the DGK inhibitor R59022 differentially affects AtDGK2 and AtDGK7 activity in vitro and alters plant growth and development.
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 approximately 80 mum inhibits root elongation and lateral root formation and reduces plant growth, indicating that DGKs play an important role in plant development.
Gómez-Merino
Fernando C
FC
University of Potsdam, Institute of Biochemistry and Biology, Karl-Liebknecht-Strasse 24-25, Haus 20, Golm/Potsdam D-14476, Germany.
Arana-Ceballos
Fernando A
FA
Trejo-Téllez
Libia Iris
LI
Skirycz
Aleksandra
A
Brearley
Charles A
CA
Dörmann
Peter
P
Mueller-Roeber
Bernd
B
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D001419:Bacteria / Q000378:metabolism; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D019852:Diacylglycerol Kinase / Q000235:genetics* / Q000502:physiology*; D004075:Diglycerides; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D018745:Genome, Plant; D005990:Glycerol / Q000031:analogs & derivatives / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D005810:Multigene Family; D009829:Oleic Acids / Q000737:chemistry; D010712:Phosphatidic Acids / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry; D018517:Plant Roots / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011744:Pyrimidinones / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors
D018076:DNA, Complementary; D003902:Detergents; D004075:Diglycerides; D004791:Enzyme Inhibitors; D009829:Oleic Acids; D010712:Phosphatidic Acids; D010940:Plant Proteins; D011744:Pyrimidinones; D011994:Recombinant Proteins; D013844:Thiazoles; D000255:Adenosine Triphosphate; C047505:R 59022; D019852:Diacylglycerol Kinase; D003545:Cysteine; D005990:Glycerol; C013965:diolein