title: Biochemical characterization of two wheat phosphoethanolamine N-methyltransferase isoforms with different sensitivities to inhibition by phosphatidic acid.
abstract: In plants the triple methylation of phosphoethanolamine to phosphocholine catalyzed by phosphoethanolamine N-methyltransferase (PEAMT) is considered a rate-limiting step in the de novo synthesis of phosphatidylcholine. Besides being a major membrane phospholipid, phosphatidylcholine can be hydrolyzed into choline and phosphatidic acid. Phosphatidic acid is widely recognized as a second messenger in stress signaling, and choline can be oxidized within the chloroplast to yield the putative osmoprotectant glycine betaine. Here we describe the cloning and biochemical characterization of a second wheat PEAMT isoform that has a four times higher specific activity than the previously described WPEAMT/TaPEAMT1 enzyme and is less sensitive to product inhibition by S-adenosyl homocysteine, but more sensitive to inhibition by phosphocholine. Both enzymes follow a sequential random Bi Bi mechanism and show mixed-type product inhibition patterns with partial inhibition for TaPEAMT1 and a strong non-competitive component for TaPEAMT2. An induction of TaPEAMT protein expression and activity is observed after cold exposure, ahead of an increase in gene expression. Our results demonstrate direct repression of in vitro enzymatic activities by phosphatidic acid for both enzymes, with TaPEAMT1 being more sensitive than TaPEAMT2 in the physiological concentration range. Other lipid ligands identified in protein-lipid overlays are phosphoinositide mono- as well as some di-phosphates and cardiolipin. These results provide new insights into the complex regulatory circuits of phospholipid biosynthesis in plants and underline the importance of head group biosynthesis in adaptive stress responses.
authors.affiliation: Environmental Biology Group, Research School of Biological Sciences, The Australian National University, G. P. O. Box 475, Canberra, Australian Capital Territory 0200, Australia.
authors.forename: Ricarda
authors.initials: R
authors.lastname: Jost
authors.forename: Oliver
authors.initials: O
authors.lastname: Berkowitz
authors.forename: John
authors.initials: J
authors.lastname: Shaw
authors.forename: Josette
authors.initials: J
authors.lastname: Masle
mesh_terms: D000064:Acclimatization; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D003080:Cold Temperature; D007074:Immunoglobulin G / Q000276:immunology; D007527:Isoenzymes; D010712:Phosphatidic Acids / Q000494:pharmacology*; D050918:Phosphatidylethanolamine N-Methyltransferase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012435:S-Adenosylhomocysteine / Q000494:pharmacology*; D014908:Triticum / Q000201:enzymology*
chemical_list: D007074:Immunoglobulin G; D007527:Isoenzymes; D010712:Phosphatidic Acids; D010716:Phosphatidylinositols; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012435:S-Adenosylhomocysteine; D050918:Phosphatidylethanolamine N-Methyltransferase