title: In Vivo and in Vitro Evidence for Biochemical Coupling of Reactions Catalyzed by Lysophosphatidylcholine Acyltransferase and Diacylglycerol Acyltransferase.
abstract: Seed oils of flax (Linum usitatissimum L.) and many other plant species contain substantial amounts of polyunsaturated fatty acids (PUFAs). Phosphatidylcholine (PC) is the major site for PUFA synthesis. The exact mechanisms of how these PUFAs are channeled from PC into triacylglycerol (TAG) needs to be further explored. By using in vivo and in vitro approaches, we demonstrated that the PC deacylation reaction catalyzed by the reverse action of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) can transfer PUFAs on PC directly into the acyl-CoA pool, making these PUFAs available for the diacylglycerol acyltransferase (DGAT)-catalyzed reaction for TAG production. Two types of yeast mutants were generated for in vivo and in vitro experiments, respectively. Both mutants provide a null background with no endogenous TAG forming capacity and an extremely low LPCAT activity. In vivo experiments showed that co-expressing flax DGAT1-1 and LPCAT1 in the yeast quintuple mutant significantly increased 18-carbon PUFAs in TAG with a concomitant decrease of 18-carbon PUFAs in phospholipid. We further showed that after incubation of sn-2-[(14)C]acyl-PC, formation of [(14)C]TAG was only possible with yeast microsomes containing both LPCAT1 and DGAT1-1. Moreover, the specific activity of overall LPCAT1 and DGAT1-1 coupling process exhibited a preference for transferring (14)C-labeled linoleoyl or linolenoyl than oleoyl moieties from the sn-2 position of PC to TAG. Together, our data support the hypothesis of biochemical coupling of the LPCAT1-catalyzed reverse reaction with the DGAT1-1-catalyzed reaction for incorporating PUFAs into TAG. This process represents a potential route for enriching TAG in PUFA content during seed development in flax.
authors.affiliation: Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada.
authors.forename: Xue
authors.initials: X
authors.lastname: Pan
authors.affiliation: Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada.
authors.forename: Guanqun
authors.initials: G
authors.lastname: Chen
authors.affiliation: National Research Council Canada, Saskatoon, Saskatchewan S7N 0W9, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Kazachkov
authors.affiliation: Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Greer
authors.affiliation: Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada.
authors.forename: Kristian Mark P
authors.initials: KMP
authors.lastname: Caldo
authors.affiliation: National Research Council Canada, Saskatoon, Saskatchewan S7N 0W9, Canada.
authors.forename: Jitao
authors.initials: J
authors.lastname: Zou
authors.affiliation: Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada. Electronic address: randall.weselake@ualberta.ca.
authors.forename: Randall J
authors.initials: RJ
authors.lastname: Weselake
mesh_terms: D008243:1-Acylglycerophosphocholine O-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D051048:Diacylglycerol O-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000235:genetics / Q000378:metabolism*; D019597:Flax / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D017343:Genes, Plant; D013379:Substrate Specificity
chemical_list: D005231:Fatty Acids, Unsaturated; D051048:Diacylglycerol O-Acyltransferase; D008243:1-Acylglycerophosphocholine O-Acyltransferase
keywords: DGAT; LPCAT; Linum usitatissimum; flax; phosphatidylcholine; phospholipid metabolism; polyunsaturated fatty acid (PUFA); triacylglycerol; triacylglycerol biosynthesis; yeast