Acyl Coenzyme A Preference of Diacylglycerol Acyltransferase from the Maturing Seeds of Cuphea, Maize, Rapeseed, and Canola

Abstract

ABSTRACrIn their seed triacylglycerols, Cuphea carthagenensis contains 62% lauric acid; maize possesses 50% linoleic acid and 30% oleic acid; rapeseed (Brassica napus L. var Dwarf Essex) has 40% erucic acid; and Canola (Brassica napus L. var Tower) holds 60% oleic acid and 23% linoleic acid. Diacylglycerol acyltransferase (EC 2.3.1.20) in the microsomal preparations fron maturing seeds of the above species were tested for their preference in using different forms of acyl coenzyme A (CoA). Lauroyl CoA, oleoyl CoA, and erucoyl CoA individually or in equimolar mixtures at increasing concentrations were added to the assay mixture containing diolein, and the formation of trinacylglycerols from the acyl groups at 24, 32, and 40°C was analyzed. The Cuphea enzyme preferred lauroyl CoA to oleoyl CoA, and was inactive on erucoyl CoA. The maize enzyme had about equal activities on oleoyl CoA and lauroyl CoA, and was inactive on erucoyl CoA. Enzymes from both rapeseed and Canola had the same pattern of acyl CoA preference, with highest activities on lauroyl CoA. The two enzymes were more active on oleoyl CoA than on erucoyl CoA at high acyl CoA concentrations (10 and 20 micromolar) at 24°C, but were more active on erucoyl CoA than on oleoyl CoA at low acyl CoA concentrations (1.36 micromolar or less) at 32 and 40°C. These findings are discussed in terms of the contribution of the enzyme to the acyl specificity in storage triacylglycerols and the implication in seed oil biotechnology.it is assumed that the in vivo pool sizes ofthe acyl CoAs determine the acyl specificity in the 3-position of the triacylglycerol.In seed oil biotechnology, one major goal is to alter the chain length of the fatty acyl moiety of the triacylglycerols (5,12,15 In an attempt to resolve part of the above unknown, we have studied the acyl CoA preference of seed diacylglycerol acyltransferase. We also would like to see if the enzyme exerts preference on specific acyl CoA, such that this preference reflects the acyl moiety in the triacylglycerol. The enzyme was obtained from four selected oil seeds which have unique and very contrasting fatty acyl moieties in the triacylglycerols. MATERIALS AND METHODSIn oil seeds, the fatty acid composition of the storage triacylglycerols is species-and variety-specific, and environmental factors such as temperature exert some modifying effects (13, 16). Within a seed species, the fatty acid composition in each of the three positions of a triacylglycerol is also largely inherited (16).In oil seeds as well as in mammalian tissues, triacylglycerols are synthesized from acyl CoA and glycerol-P via three different acyltransferases (1,13,16,17). The first two acyltransferases (glycerol-P acyltransferase and lysophosphatidic acid acyltransferase) possess some acyl CoA preference, such that their specificities as well as the in vivo pool sizes of acyl CoAs produce the observed positional acyl specificity in the triacylglycerol (1,17

Triacylglycerol Bioassembly in Microspore-Derived Embryos of Brassica napus L. cv Reston

“…The absolute activity expressed in this fraction was greater when G-3-P, rather than DAG, was supplied as acceptor (Table V). In general, the activities recorded in the presence of DAG acceptors in this study were comparable to 22:1-CoA:DGAT activities recorded in microsomal preparations from zygotic embryos of high 22:1 cvs of B. napus (8,13).…”

Isolation of a Gene Encoding a 1,2-Diacylglycerol-sn-acetyl-CoA Acetyltransferase from Developing Seeds of Euonymus alatus

“…Triacylglycerol synthesis has been described in developing seed extracts from various plant species using medium-chain acyl-CoAs and/or medium-chain diacylglycerols in DGAT as-says (17)(18)(19)(20). Frequently, the substrate specificity and selectivity of these DGATs are fairly broad.…”

Lysophosphatidate Acyltransferase Activities in the Microsomes from Palm Endosperm, Maize Scutellum, and Rapeseed Cotyledon of Maturing Seeds

“…In oil seeds, the first and third acyltransferase enzymes, glycerol-3-P AT and DAG-AT, were studied by various workers (3,4,7,10,12,15). These studies show that, in general, glycerol-3-P AT preferentially uses palmitoyl CoA over C-18 CoAs (7,8,10).…”

“…Our recent studies suggest that these unusual fatty acids may not be readily utilized for TAG synthesis in seeds where they are not normally found. In our studies, we tested the ability of the enzymes from three selected oil seeds, each containing a different abundant fatty acid in their storage TAG (palm or Cuphea, Cl 2; maize, Cl 8:1, rapeseed C22: 1), to utilize those fatty acids of unusual chain length for TAG synthesis in vitro (3,22). The results show that while DAG-AT from Cuphea, maize, and rapeseed can utilize lauroyl CoA and oleoyl CoA for TAG synthesis, only the rapeseed enzyme has activity with erucoyl CoA (3).…”

“…In our studies, we tested the ability of the enzymes from three selected oil seeds, each containing a different abundant fatty acid in their storage TAG (palm or Cuphea, Cl 2; maize, Cl 8:1, rapeseed C22: 1), to utilize those fatty acids of unusual chain length for TAG synthesis in vitro (3,22). The results show that while DAG-AT from Cuphea, maize, and rapeseed can utilize lauroyl CoA and oleoyl CoA for TAG synthesis, only the rapeseed enzyme has activity with erucoyl CoA (3). Also, when radioactive glycerol-3-P was incubated with palm, maize, or rapeseed microsomes in the presence of nonradioactive acyl CoA, all three preparations synthesized radioactive LPA, PA, DAG, and TAG from oleoyl CoA (22).…”