Arabidopsis ATDGK7, the smallest member of plant diacylglycerol kinases, unique biochemical features and saturates at low substrate.

Fernando C. Gómez-Merino, Fernando A. Arana-Ceballos, Libia Iris Trejo-Téllez, Aleksandra Skirycz, Charles A. Brearley, Peter Dörmann, Bernd Mueller-Roeber

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Abstract

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 ~80 µm inhibits root elongation and lateral root formation and reduces plant growth, indicating that DGKs play an important role in plant development.
Original languageEnglish
Pages (from-to)34888-34899
Number of pages12
JournalJournal of Biological Chemistry
Volume280
DOIs
Publication statusPublished - 14 Oct 2005

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