Biosynthesis of myristate in an aphid: Involvement of a specific acylthioesterase

Abstract

Approximately 70% of the total fatty acids of the pea aphid, Acyrthosiphon pisum, is myristate. The fatty acids of the phospholipid fraction consist of over 80% 18-carbon saturated and unsaturated fatty acids, whereas myristate accounts for almost 90% of the fatty acids of the triacylglycerol fraction. In vivo studies showed that [1- 14C]acetate was incorporated into both saturated (primarily myristate) and monounsaturated fatty acids, with the 14-carbon fatty acid incorporated almost exclusively into the triacylglycerol fraction and the 16- and 18-carbon fatty acids preferentially incorporated into the phospholipid fraction. A 105,000 g particle-free supernatant preparation incorporated radiolabel from [ 3H]acetyl-coenyzme A (CoA) primarily into myristate at low malonyl-CoA concentrations, and primarily into 16- and 18-carbon fatty acids at high malonyl-CoA concentrations. Using low malonyl-CoA concentrations, [ 3H]acetyl-CoA was preferentially incorporated into myristate at low ionic stengths, and into 16- and 18-carbon fatty acids at high ionic strengths. Sepharose 6B column chromatography was used to partially purify the fatty acid synthetase (FAS). The partially purified FAS produced 16- and 18-carbon fatty acids exclusively, regardless of malonyl-CoA concentrations or ionic strengths. However, when a separate heat-labile fraction from the Sepharose 6B column was added to the partially purified FAS, it restored the ability of the FAS to produce myristate. Adding larger amounts of this fraction to the partially purified FAS resulted in the formation of a higher percentage of myristate. This fraction contained thioesterase activity, and was partially resolved into two thioesterase activities by Sephadex G-100 column chromatography. The later eluting thioesterase, but not the earlier eluting one, was responsible for chain termination at 14 carbons. This thioesterase readily cleaved 18-, 16-, and 14-carbon acyl-CoA's, but had a much lower specificity for a C 12 CoA. This later eluting thioesterase also restored activity to FAS treated with phenylmethanesulfonyl fluoride. These results suggest the presence of an acylthioesterase in the aphid which releases the growing acyl chain from the FAS when it reaches 14 carbons.