Abstract
Lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23) is an evolutionarily conserved key enzyme in the Lands cycle that catalyzes acylation of lysophosphatidylcholine (LPC) to produce phosphatidylcholine (PC), the main phospholipid in cellular membranes. In this study, three LPCAT genes from sunflower were identified and the corresponding proteins characterized. These HaLPCAT genes encoded functionally active enzymes that were able to complement a deficient yeast mutant. Moreover, enzymatic assays were carried out using microsomal preparations of the yeast cells. When acyl specificities were measured in the forward reaction, these enzymes exhibited a substrate preference for unsaturated acyl-CoAs, especially for linolenoyl-CoA, while in the reverse reaction, linoleoyl or linolenoyl acyl groups were transferred from PC to acyl-CoA to a similar extent. Expression levels of LPCAT genes were studied revealing distinct tissue-specific expression patterns. In summary, this study suggests that the combined forward and reverse reactions catalyzed by sunflower LPCATs facilitate acyl-exchange between the sn-2 position of PC and the acyl-CoA pool. Sunflower LPCATs displayed different characteristics, which could point to different functionalities, favoring the enrichment of seed triacylglycerols (TAGs) with polyunsaturated fatty acid (PUFA).
Highlights
Oleaginous seeds accumulate TAGs as reserve material which is used to feed the embryo during germination (Lu et al, 2011; Chapman and Ohlrogge, 2012)
Three genes coding for sunflower LPCAT were cloned from cDNA obtained from developing sunflower seed mRNA by PCR using specific primers (Supplementary Table S1)
They displayed a high level of identity with very few gaps, even though a large taxonomic range of species was included (Supplementary Figure S1). This pointed to a high degree of conservation among these enzymes along the period of evolution of the species investigated. When these proteins were characterized using the Conserved Domain Database (CDD) (Marchler-Bauer et al, 2017), they were classified within the membrane-bound O-acyltransferase (MBOAT) family, a group of membranebound enzymes that catalyzes the transfer of acyl-moieties to various acyl acceptors
Summary
Oleaginous seeds accumulate TAGs as reserve material which is used to feed the embryo during germination (Lu et al, 2011; Chapman and Ohlrogge, 2012). LPCAT enzymes catalyze the reacylation of LPC using acyl moieties coming from the acyl-CoA pool (Stymne and Stobart, 1987; Bates and Browse, 2012) Together with phospholipases, these enzymes operate within the Lands cycle (Lands, 1965; Wang et al, 2012) to help maintain an active and continuous turnover of fatty acids in cellular membranes. A later work on soybean developing embryos showed that TAG synthesis in oil accumulating tissues involves different pools of DAG and PC It was experimental evidence of a large bulk PC pool created by acyl edition through the action of LPCATs coexisting with a smaller active PC pool coming from de novo DAG synthesis. The role of LPCAT enzymes in the transfer of PUFAs from PC to TAG was reinforced by results published by Bates et al (2012)
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