A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX 4 D acyltransferase motif at the N-terminal end and a lipid binding motif VX 2 GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid. Triacylglycerol (TAG),3 an acyl ester glycerol, is the most efficient storage form of energy in eukaryotic cells (1, 2). In plants, TAG is the major component of the seed oils and is mainly deposited during seed and fruit development. Plant oils have a tremendous socioeconomic value in the food industry as well as in the production of lubricants and biofuels. They are also becoming increasingly important raw materials in the petrochemical industry as a substitute for non-renewable crude oil reserves (3). Thus, numerous conventional and molecular genetic strategies have been explored in attempts to increase the TAG content and modify the fatty acid composition of the plant seed oils. Isolation of the genes involved in oil biosynthesis offer exciting opportunities for overexpressing the desired gene of interest so as to redesign the plant oil metabolism for greater yields (3).TAG is usually synthesized by the sequential incorporation of acyl groups to glycerol 3-phosphate by substrate-specific acyltransferases to give lysophosphatidic acid and phosphatidic acid, respectively (4, 5). The synthesized phosphatidic acid is dephosphorylated to diacylglycerol (DAG). The last step in this pathway is catalyzed by acyl-CoA:DAG acyltransferase (DGAT; EC 2.3.1.20) that transfers an acyl group to DAG to form the TAG. DAG lies at the branch point between phospholipid and storage lipid biosynthesis. Therefore, much research has been focused on the acyl-CoA-dependent reaction catalyzed by the DAG acyltransferase, which is an integral endoplasmic reticulum protein (6) and has also been shown to be present in the oil bodies (7) and plastids (8). Several lines of evidence suggest that the TAG biosynthesis and DGAT activity are ...
Monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, the precursor of triacylglycerol biosynthesis and an important signaling molecule. Here, we describe the isolation and characterization of the peanut (Arachis hypogaea) MGAT gene. The soluble enzyme utilizes invariant histidine-62 and aspartate-67 residues of the acyltransferase motif for its MGAT activity. A sequence analysis revealed the presence of a hydrolase (GXSXG) motif, and enzyme assays revealed the presence of monoacylglycerol (MAG) and lysophosphatidylcholine (LPC) hydrolytic activities, indicating the bifunctional nature of the enzyme. The overexpression of the MGAT gene in yeast (Saccharomyces cerevisiae) caused an increase in triacylglycerol accumulation. Similar to the peanut MGAT, the Arabidopsis (Arabidopsis thaliana) homolog (At1g52760) also exhibited both acyltransferase and hydrolase activities. Interestingly, the yeast homolog lacks the conserved HX 4 D motif, and it is deficient in the acyltransferase function but exhibits MAG and LPC hydrolase activities. This study demonstrates the presence of a soluble MGAT/hydrolase in plants. The predicted three-dimensional homology modeling and substrate docking suggested the presence of two separate substrate (MAG and LPC)-binding sites in a single polypeptide. Our study describes a soluble bifunctional enzyme that has both MGAT and hydrolase functions.
A soluble diacylglycerol acyltransferase is involved in triacylglycerol biosynthesis in the oleaginous yeast Rhodotorula glutinis The biosynthesis of triacylglycerol (TAG) occurs in the microsomal membranes of eukaryotes. Here, we report the identification and functional characterization of diacylglycerol acyltransferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in Rhodotorula glutinis. Both a full-length and an N-terminally truncated cDNA clone of a single gene were isolated from R. glutinis. The DGAT activity of the protein encoded by RgDGAT was confirmed in vivo by the heterologous expression of cDNA in a Saccharomyces cerevisiae quadruple mutant (H1246) that is defective in TAG synthesis. RgDGAT overexpression in yeast was found to be capable of acylating diacylglycerol (DAG) in an acyl-CoA-dependent manner. Quadruple mutant yeast cells exhibit growth defects in the presence of oleic acid, but wild-type yeast cells do not. In an in vivo fatty acid supplementation experiment, RgDGAT expression rescued quadruple mutant growth in an oleate-containing medium. We describe a soluble acyl-CoA-dependent DAG acyltransferase from R. glutinis that belongs to the DGAT3 class of enzymes. The study highlights the importance of an alternative TAG biosynthetic pathway in oleaginous yeasts.
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