<i>Claviceps purpurea</i>, a fungal pathogen, of ergot diseases in agriculturally important cereal crops, produces high levels of glycerides containing ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. A fatty acid hydroxylase (CpFAH) involved in the biosynthesis of ricinoleic acid was recently identified from <i>C. purpurea</i>. This research describes the biochemical characterization of a type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of this fatty acid into triglycerides from <i>C. purpurea</i>. Expression of CpDGAT2 in a quadruple mutant <i>Saccharomyses cerevisiae</i> H1246, in which all four triacylglycerol (TG) biosynthesis genes (DGA1, LOR1, ACAT1 and ACAT2) were disrupted, restored the ability of the mutant to synthesize TGs <i>in vivo</i>. <i>In vitro</i> enzymatic assays of microsomal preparations of the transformants indicated that CpDGAT2 preferentially use ricinoleic acid over linoleic acid, oleic acid and linolenic acids as acyl donor, and 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as acyl acceptor. CpDGAT2 did not show any activities for the formation of wax esters and estolides when 1-hexadecanol and triricinolein were used as acyl acceptors. Co-expression of CpFAH and CpDGAT2 in yeast resulted in increased accumulation of ricinoleic acids compared to expression of CpFAH along with the yeast native DGAT2 (ScDGA1) or expression of CpFAH alone. Northern blot analysis indicated that CpFAH is solely expressed in sclerotium cells and no transcripts of this gene were detected in mycelium and conidium cells. CpDGAT2 is more widely expressed in cell types examined except for conidiospores where the expression is low. The highest expression of CpDGAT2 was detected in 20 day-old sclerotium cells where the highest levels of ricinoleate glycerides are accumulated. Collectively, these data indicate CpDGAT2 and CpFAH are two key enzymes coordinating the biosynthesis and bioassembly of ricinoleic acid in <i>C. purpurea</i>.

<i>Claviceps purpurea</i>, a fungal pathogen, of ergot diseases in agriculturally important cereal crops, produces high levels of glycerides containing ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. A fatty acid hydroxylase (CpFAH) involved in the biosynthesis of ricinoleic acid was recently identified from <i>C. purpurea</i>. This research describes the biochemical characterization of a type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of this fatty acid into triglycerides from <i>C. purpurea</i>. Expression of CpDGAT2 in a quadruple mutant <i>Saccharomyses cerevisiae</i> H1246, in which all four triacylglycerol (TG) biosynthesis genes (DGA1, LOR1, ACAT1 and ACAT2) were disrupted, restored the ability of the mutant to synthesize TGs <i>in vivo</i>. <i>In vitro</i> enzymatic assays of microsomal preparations of the transformants indicated that CpDGAT2 preferentially use ricinoleic acid over linoleic acid, oleic acid and linolenic acids as acyl donor, and 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as acyl acceptor. CpDGAT2 did not show any activities for the formation of wax esters and estolides when 1-hexadecanol and triricinolein were used as acyl acceptors. Co-expression of CpFAH and CpDGAT2 in yeast resulted in increased accumulation of ricinoleic acids compared to expression of CpFAH along with the yeast native DGAT2 (ScDGA1) or expression of CpFAH alone. Northern blot analysis indicated that CpFAH is solely expressed in sclerotium cells and no transcripts of this gene were detected in mycelium and conidium cells. CpDGAT2 is more widely expressed in cell types examined except for conidiospores where the expression is low. The highest expression of CpDGAT2 was detected in 20 day-old sclerotium cells where the highest levels of ricinoleate glycerides are accumulated. Collectively, these data indicate CpDGAT2 and CpFAH are two key enzymes coordinating the biosynthesis and bioassembly of ricinoleic acid in <i>C. purpurea</i>.

Triacylglycerols are the predominant storage form of energy in eukaryotes. As obesity has become a worldwide problem and excessive accumulation of triacylglycerols in adipose tissue causes obesity, enzymes catalyzing the synthesis of triacylglycerols are of great interest. Acyl CoA:diacylglycerol acyltransferase (DGAT), including the isoforms DGAT1 and DGAT2, catalyze the final and committed step in triacylglycerol synthesis. Proteins that physically interact with DGAT1 may provide information regarding the metabolic role of DGAT1. We chose HEK-293T cell line to express DGAT1 and used mass spectrometry to identify proteins that co-immunoprecipitated with DGAT1. We confirmed that DGAT2 and ACAT1 did interact with DGAT1. The interaction of DGAT1 with DGAT2 appeared to interrupt the synthesis of triacylglycerol since the co-expression of DGAT1 and DGAT2 was expected to increase triacylglycerol synthesis. This implied that DGAT1 and DGAT2 might serve different functional roles. On the other hand, DGAT1 overexpression may increase the synthesis of cholesterol esters that was the product of ACAT1. Additionally, ACAT1 overexpression did increase triacylglycerol synthesis and ACAT1 disruption by siRNA did decrease triacylglycerol synthesis. Our findings indicated that DGAT1 and ACAT1 might be involved in the same lipid-synthesizing protein complex.

No description available.

Acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20, DGAT or DAGAT) is a membrane protein found mainly in the endoplasmic reticulum (ER). It catalyzes the final step in the biosynthesis of triacylglyerol (TAG or TG), which is the principal repository of fatty acids for energy utilization and membrane formation. Several lines of evidence have indicated that DGAT has a substantial effect on carbon flux into TAG. DGAT has at least two discrete family members (DGAT1 and DGAT2) with different physiological roles. High-resolution structures of both DGATs, however, are absent due to difficulties in purification. In order to gain insight into structural and functional relationships of DGATs, a functional DGAT2 protein from the yeast Saccharomyces cerevisiae (ScDGAT2, also known as Dgalp) was selected. The structural and functional role of cysteine residues in ScDGAT2 was studied using site-directed mutagenesis (SDM) in combination with chemical modification. Although ScDGAT2 is susceptible to thiol-modifying reagents, none of the cysteines are essential for the catalytic activity or involved in structure support though disulfide linkages. Inhibition of DGAT activity by thiol-specific modification was localized to cysteine314, which is in the proximity of a highly conserved motif in DGAT2s. Thus, cysteine314 may reside in a crucial position near a possible active site or related to proper protein folding. The functional importance and topological orientation of signature motifs in ScDGAT2 were also studied using the same methods. Both the N- and C-termini of ScDGAT2 are oriented toward the cytosol. A highly conserved motif, 129YFP131, and a hydrophilic segment exclusive to ScDGAT2, reside in the ER and play essential roles in enzyme catalysis. In addition, the strongly conserved H195, which may be part of the active site of DGAT2, is likely embedded in the membrane. Although ScDGAT2 has a topology similar to that of murine DGAT2, there are striking differences which suggest that the topological organization of DGAT2 is not ubiquitously conserved. / Plant Science

No description available.

No description available.

The neutral lipid triacylglycerol (TAG) is the main storage lipid in plants. When stored in seeds, TAG provides the carbon and energy source during germination. There is significant human nutritional demand for vegetable oil, but its use in production of renewable biomaterials and fuels has intensified the need to increase oil production. In plants, the final and committed step in TAG biosynthesis is catalyzed by diacylglycerol acyltransferases (DGAT) and/or a phospholipid: diacylglycerol acyltransferases (PDAT). Both DGAT and PDAT contribute to seed TAG biosynthesis in an independent or overlapping manner, depending on the species. However, in nonseed tissues such as mesocarp of avocado, the regulation of TAG biosynthesis is not well-studied. Based on the transcriptome data of Persea americana it is hypothesized that both DGAT and PDAT are likely to catalyze the conversion of diacylglycerol to TAG. In this study, putative DGAT1 and DGAT2 were identified and comprehensive in silico analyses were conducted to determine the respective start codons, full-length coding sequences, transmembrane domains, predicted protein structures and phylogenetic relationships with other known DGATs. These data reveal that the putative DGATs of a basal angiosperm species retain features that are conserved not only among angiosperms but also other eukaryotes. For further biochemical characterization, the avocado DGATs were expressed in a TAGdeficient yeast strain and lipotoxicity rescue assays were conducted. The complementation of this yeast strain confirmed enzyme activity and supported the possible role of both avocado DGATs in TAG biosynthesis. Future studies will be focused on determining the substrate specificity of DGAT and its role, relative to PDATs in TAG biosynthesis in avocado mesocarp.

The avocado mesocarp contains up to 60-70% oil by dry weight where triacylglycerol (TAG) is the major constituent. This neutral lipid, TAG is utilized by plants for the carbon and energy source when stores in seed tissue. There is significant human nutritional demand for vegetable oil, but its use in production of renewable biomaterials and fuels has intensified the need to increase oil production. In plants, the final and committed step in TAG biosynthesis is catalyzed by diacylglycerol acyltransferases (DGAT) and/or a phospholipid: diacylglycerol acyltransferases (PDAT). Both DGAT and PDAT contribute to seed TAG biosynthesis in an independent or overlapping manner, depending on the species. However, the regulation of TAG biosynthesis is not well-studied in nonseed tissues such as mesocarp of avocado. Based on the transcriptome data of Persea americana it is hypothesized that both DGAT and PDAT are likely to catalyze the conversion of diacylglycerol to TAG. In this study, putative DGAT1 and DGAT2 were identified and comprehensive in silico analyses were conducted to determine the respective start codons, full-length coding sequences, transmembrane domains, predicted protein structures and phylogenetic relationships with other known DGATs. These data reveal that the putative DGATs of a basal angiosperm species retain features that are conserved not only among angiosperms but also other eukaryotes. For further functional analysis, the avocado DGATs were expressed in H1246, a TAG-deficient yeast strain and lipotoxicity rescue assays were conducted. The complementation of this yeast strain confirmed enzyme activity and supported the possible role of both avocado DGATs in TAG biosynthesis. Future studies will be focused on determining the substrate specificity of DGAT and its role, relative to PDATs in TAG biosynthesis in avocado mesocarp.