Identification, purification, and characterization of monoacylglycerol acyltransferase from developing peanut cotyledons

Biosynthesis of diacylglycerols in plants occurs mainly through the acylati on of lysophosphatidic acid in the microsomal membranes. Here we describe t he first identification of diacylglycerol biosynthetic activity in the solu ble fraction of developing oilseeds. This activity was NaF-insensitive and acyl-CoA-dependent Diacylglycerol formation was catalyzed by monoacylglycer ol (MAG) acyltransferase (EC 2.3.1.22) that transferred an acyl moiety from acyl-CoA to MAG. The enzyme was purified by successive chromatographic sep arations on octyl-Sepharose, blue-Sepharose, Superdex-75, and palmitoyl-CoA -agarose to apparent homogeneity from developing peanut (Arachis hypogaea) cotyledons, The enzyme was purified to 6,608-fold with the final. specific activity of 15.86 nmol min(-1) mg(-1). The purified enzyme was electrophore tically homogeneous, and its molecular mass was 43,000 daltons, The purifie d MAG acyltransferase was specific for MAG and did not utilize any other ac yl acceptor such as glycerol, glycerol-3-phosphate, lysophosphatidic acid, and lysophosphatidylcholine. The K-m values for 1-palmitoylglycerol and 1-o leoylglycerol were 16.39 and 5.65 phn, respectively. The K-m values for 2-m onoacylglycerols were 2- to 4-fold higher than that of the corresponding 1- monoacylglycerol, The apparent K-m values for palmitoyl-, stearoyl-, and ol eoyl-CoAs were 17.54, 25.66, and 9.35 muM, respectively. Fatty acids, phosp holipids, and sphingosine at low concentrations stimulated the enzyme activ ity. The identification of MAG acyltransferase in oilseeds suggests the pre sence of a regulatory link between signal transduction and synthesis of com plex lipids in plants.