ABSTRACT
Acyltransferase enzymes have been reported as useful biotechnological tools in order to increase oil yield and modify fatty acid composition. Macadamia species are able to accumulate unusually high levels of palmitoleic acid that besides oleic acid amounts to over 80% of monounsaturated fatty acids in the seed oil. In this work, a gene encoding a type 1 acyl-CoA:diacylglycerol acyltransferase (DGAT1) was cloned from M. tetraphylla. DGAT activity of the protein encoded by MtDGAT1 was confirmed by heterologous expression in a yeast mutant. Fatty acid composition of triacylglycerols synthesized by MtDGAT1 was compared to that of DGAT1 enzymes from Arabidopsis and Echium, with the results suggesting a substrate preference for monounsaturated over polyunsaturated fatty acids. Characteristics of MtDGAT1 may contribute to biochemical mechanisms determining the particular fatty acid composition of Macadamia oil and also indicate the possibility of using this enzyme in biotechnological approaches where a reduction of polyunsaturated fatty acids in the oil is desired.
Subject(s)
Cloning, Molecular , Diacylglycerol O-Acyltransferase/chemistry , Diacylglycerol O-Acyltransferase/genetics , Macadamia/enzymology , Plant Proteins/chemistry , Plant Proteins/genetics , Triglycerides/chemistry , Amino Acid Sequence , Diacylglycerol O-Acyltransferase/metabolism , Enzyme Stability , Gene Expression , Macadamia/chemistry , Macadamia/genetics , Molecular Sequence Data , Nuts/chemistry , Nuts/enzymology , Nuts/genetics , Plant Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Alignment , Triglycerides/metabolismABSTRACT
Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23) catalyzes acylation of lysophosphatidylcholine (lysoPtdCho) to produce phosphatidylcholine (PtdCho), the main phospholipid in cellular membranes. This reaction is a key component of the acyl-editing process, involving recycling of the fatty acids (FA) mainly at the sn-2 position of PtdCho. Growing evidences indicate that the LPCAT reaction controls the direct entry of newly synthesized FA into PtdCho and, at least in some plant species, it has an important impact on the synthesis and composition of triacylglycerols. Here we describe the molecular characterization of the single LPCAT gene found in the genome of Ricinus communis (RcLPCAT) that is homologous to LPCAT genes of the MBOAT family previously described in Arabidopsis and Brassica. RcLPCAT is ubiquitously expressed in all organs of the castor plant. Biochemical properties have been studied by heterologous expression of RcLPCAT in the ale1 yeast mutant, defective in lysophospholipid acyltransferase activity. RcLPCAT preferentially acylates lysoPtdCho against other lysophospholipids (lysoPL) and does not discriminates the acyl chain in the acceptor, displaying a strong activity with alkyl lysoPL. Regarding the acyl-CoA donor, RcLPCAT uses monounsaturated fatty acid thioesters, such as oleoyl-CoA (18:1-CoA), as preferred donors, while it has a low activity with saturated fatty acids and shows a poor utilization of ricinoleoyl-CoA (18:1-OH-CoA). These characteristics are discussed in terms of a possible role of RcLPCAT in regulating the entry of FA into PtdCho and the exclusion from the membranes of the hydroxylated FA.
Subject(s)
1-Acylglycerophosphocholine O-Acyltransferase/genetics , Lysophosphatidylcholines/metabolism , Phosphatidylcholines/metabolism , Plant Proteins/genetics , Ricinus communis/chemistry , 1-Acylglycerophosphocholine O-Acyltransferase/classification , 1-Acylglycerophosphocholine O-Acyltransferase/metabolism , Acyl Coenzyme A/metabolism , Amino Acid Sequence , Arabidopsis/enzymology , Arabidopsis/genetics , Brassica/enzymology , Brassica/genetics , Ricinus communis/enzymology , Ricinus communis/genetics , Enzyme Assays , Fatty Acids/metabolism , Kinetics , Molecular Sequence Data , Phylogeny , Plant Proteins/classification , Plant Proteins/metabolism , Recombinant Proteins/classification , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
The multigene family encoding proteins related to lysophosphatidyl-acyltransferases (LPATs) has been analyzed in the castor plant Ricinus communis. Among them, two genes designated RcLPAT2 and RcLPATB, encoding proteins with LPAT activity and expressed in the developing seed, have been cloned and characterized in some detail. RcLPAT2 groups with well characterized members of the so-called A-class LPATs and it shows a generalized expression pattern in the plant and along seed development. Enzymatic assays of RcLPAT2 indicate a preference for ricinoleoyl-CoA over other fatty acid thioesters when ricinoleoyl-LPA is used as the acyl acceptor, while oleoyl-CoA is the preferred substrate when oleoyl-LPA is employed. RcLPATB groups with B-class LPAT enzymes described as seed specific and selective for unusual fatty acids. However, RcLPATB exhibit a broad specificity on the acyl-CoAs, with saturated fatty acids (12:0-16:0) being the preferred substrates. RcLPATB is upregulated coinciding with seed triacylglycerol accumulation, but its expression is not restricted to the seed. These results are discussed in the light of a possible role for LPAT isoenzymes in the channelling of ricinoleic acid into castor bean triacylglycerol.
