Rodrigo M. P. Siloto

Acyl-CoA:diacylglycerol acyltransferase: molecular biology, biochemistry and biotechnology.

Triacylglycerol (TG) is a storage lipid which serves as an energy reservoir and a source of signalling molecules and substrates for membrane biogenesis. TG is essential for many physiological processes and its metabolism is widely conserved in nature. Acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the final step in the sn-glycerol-3-phosphate pathway leading to TG. DGAT activity resides mainly in two distinct membrane bound polypeptides, known as DGAT1 and DGAT2 which have been identified in numerous organisms. In addition, a few other enzymes also hold DGAT activity, including the DGAT-related acyl-CoA:monoacylglycerol acyltransferases (MGAT). Progress on understanding structure/function in DGATs has been limited by the lack of detailed three-dimensional structural information due to the hydrophobic properties of theses enzymes and difficulties associated with purification. This review examines several aspects of DGAT and MGAT genes and enzymes, including current knowledge on their gene structure, expression pattern, biochemical properties, membrane topology, functional motifs and subcellular localization. Recent progress in probing structural and functional aspects of DGAT1 and DGAT2, using a combination of molecular and biochemical techniques, is emphasized. Biotechnological applications involving DGAT enzymes ranging from obesity therapeutics to oilseed engineering are also discussed.

Functional and topological analysis of yeast acyl-CoA:Diacylglycerol acyltransferase 2, an endoplasmic reticulum enzyme essential for triacylglycerol biosynthesis

Acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20) is a membrane protein present mainly in the endoplasmic reticulum. It catalyzes the final and committed step in the biosynthesis of triacylglycerol, which is the principal repository of fatty acids for energy utilization and membrane formation. Two distinct family members of acyl-CoA:diacylglycerol acyltransferase, known as DGAT1 and DGAT2, have been characterized in different organisms, including mammals, fungi, and plants. In this study, we characterized the functional role and topological orientation of signature motifs in yeast (Saccharomyces cerevisiae) DGAT2 using mutagenesis in conjunction with chemical modification. Our data provide evidence that both the N and C termini are oriented toward the cytosol and have different catalytic roles. A highly conserved motif, 129YFP131, and a hydrophilic segment exclusive to yeast DGAT2 reside in a long endoplasmic reticulum luminal loop following the first transmembrane domain and play an essential role in enzyme catalysis. In addition, the strongly conserved His195 within the motif HPHG, which may play a role in the active site of DGAT2, is likely embedded in the membrane. These results indicate some similarities to the topology model of murine DGAT2 but also reveal striking differences suggesting that the topological organization of DGAT2 is not ubiquitously conserved.

Simple Methods to Detect Triacylglycerol Biosynthesis in a Yeast-Based Recombinant System

Standard methods to quantify the activity of triacylglycerol (TAG) synthesizing enzymes DGAT and PDAT (TAG-SE) require a sensitive but rather arduous laboratory assay based on radio-labeled substrates. Here we describe two straightforward methods to detect TAG production in baker’s yeast Saccharomyces cerevisiae. First we demonstrate that a quadruple knockout yeast strain deficient in storage lipids has a reduced growth rate in a medium supplemented with fatty acids. This phenotype is rescued by restoring TAG biosynthesis and can be thus used to select yeast cells expressing a recombinant TAG-SE. In the second method, the activity of the recombinant enzyme is measured in a fluorescent in situ assay using Nile red dye that is specific for neutral lipids. Correlation between Nile red fluorescence and enzyme activity is demonstrated with several mutants of a TAG synthesizing enzyme. This yeast live-cell-based assay is rapid, inexpensive, sensitive, and is amenable to high-throughput applications. The methods can be used for a variety of applications such as isolation of novel genes, directed evolution, gene-specific drug screening and will facilitate novel approaches in the research of TAG-SE.

Directed evolution of acyl-CoA:diacylglycerol acyltransferase: Development and characterization of Brassica napus DGAT1 mutagenized libraries

Metabolic flux to triacylglycerol (TAG) may be limited by the level of acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) activity. In some species, this enzyme also appears to play a role in the channeling of specific fatty acyl moieties into TAG. The objective of this work is to implement a directed evolution approach to enhance the catalytic efficiency of type-1 DGAT from Brassica napus (BnDGAT1). We generated randomly mutagenized libraries of BnDGAT1 in a yeast expression vector using error-prone PCR. The mutagenized libraries were used to transform a Saccharomyces cerevisiae strain devoid of neutral lipid biosynthesis and analyzed using a high-throughput screening (HTS) system. The HTS, recently developed for this purpose, consisted of a positive selection of clones expressing active DGAT mutants followed by quantification of DGAT activity by fluorescence detection of TAG in yeast cells. The initial results indicated that the positive selection system efficiently eliminated DGAT mutants lacking enzyme activity. Screening of 1528 selected mutants revealed that some DGAT clones had enhanced ability to synthesize TAG in yeast. This was confirmed by analysis of individual clones that could carry mutations resulting in an increased catalytic efficiency. The directed evolution approach could lead to the development of an improved plant DGAT1 for increasing seed oil content in oleaginous crops.

Flax (Linum usitatissimum L.)

Flax is a temperate industrial oilseed crop grown primarily in Canada, China, and Russia. Flax is a diploid, autogamous species, and breeding follows traditional methods, enhanced by mutation breeding and the use of genetic markers. Recently available flax genomic resources may hasten the achievement of breeding objectives: increased yield, shorter time to mature, disease resistance, and seed oil quality. Flax oil is unique because it is contains up to 64% α-linolenic acid (ALA). ALA polymerizes rapidly with exposure to oxygen and is therefore useful in varnishes, inks, linoleum, and other traditional industrial applications. ALA is also a metabolic precursor to ω-3 polyunsaturated fatty acids (PUFAs), which have positive effects with respect to cardiovascular health and inflammatory diseases, as well as anticancer properties. In addition to ALS, flax contains antioxidants and phytosterols that may increase health benefits. Flax is being used as a functional food ingredient for humans and animal feed to increase the ω-3 fatty acids in eggs and meat. Considerable progress has been made in understanding and enhancing the metabolic pathways leading to ALA and PUFA synthesis in flax. Further research investment in this niche crop will increase the scope of utilization for industrial, food and feed oil, and fiber byproducts.

Identification of a Pair of Phospholipid:Diacylglycerol Acyltransferases from Developing Flax (Linum usitatissimum L.) Seed Catalyzing the Selective Production of Trilinolenin

Background: Triacylglycerol (TAG) can be formed via an acyl-CoA-dependent or acyl-CoA-independent pathway. Results: Overexpressing particular flax phospholipid:diacylglycerol acyltransferase (PDAT) genes in yeast and Arabidopsis resulted in an enhanced proportion of α-linolenic acid (ALA) in TAG. Conclusion: Certain PDATs have the unique ability to efficiently channel ALA into TAG. Significance: The identified PDATs will benefit future projects aimed at producing oils with enhanced polyunsaturated fatty acid content. The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3cisΔ9,12,15) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce ∼57% ALA in triacylglycerol (TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1 and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties. The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty acids in oilseeds for food and industrial applications.

Lipins from plants are phosphatidate phosphatases that restore lipid synthesis in a pah1Δ mutant strain of Saccharomyces cerevisiae

The identification of the yeast phosphatidate phosphohydrolase (PAH1) gene encoding an enzyme with phosphatidate phosphatase (PAP; 3‐sn‐phosphatidate phosphohydrolase, EC 3.1.3.4) activity led to the discovery of mammalian Lipins and subsequently to homologous genes from plants. In the present study, we describe the functional characterization of Arabidopsis and Brassica napus homologs of PAH1. Recombinant expression studies confirmed that homologous PAHs from plants can rescue different phenotypes exhibited by the yeast pah1Δ strain, such as temperature growth sensitivity and atypical neutral lipid composition. Using this expression system, we examined the role of the putative catalytic motif DXDXT and other conserved residues by mutational analysis. Mutants within the carboxy‐terminal lipin domain displayed significantly decreased PAP activity, which was reflected by their limited ability to complement different phenotypes of pah1Δ. Subcellular localization studies using a green fluorescent protein fusion protein showed that Arabidopsis PAH1 is mostly present in the cytoplasm of yeast cells. However, upon oleic acid stimulation, green fluorescent protein fluorescence was predominantly found in the nucleus, suggesting that plant PAH1 might be involved in the transcriptional regulation of gene expression. In addition, we demonstrate that mutation of conserved residues that are essential for the PAP activity of the Arabidopsis PAH1 enzyme did not impair its nuclear localization in response to oleic acid. In conclusion, the present study provides evidence that Arabidopsis and B. napus PAHs restore lipid synthesis in yeast and that DXDXT is a functional enzymic motif within plant PAHs.

Antisense suppression of type 1 diacylglycerol acyltransferase adversely affects plant development in Brassica napus

Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-coenzyme A (CoA) dependent acylation of sn-1,2-diacylglycerol to form triacylglycerol in the terminal step of seed oil formation. Previous work has suggested that the level of DGAT activity may have a substantial effect on the flow of carbon into triacylglycerol, implying that the enzyme may represent a promising target for seed oil modification through biotechnological approaches. In the current study, Brassica napus DH12075 was transformed with an antisense type 1 DGAT construct, resulting in a reduction in DGAT1 gene expression, total DGAT activity and seed oil content. In addition, reduced seed yield and germination rates were observed along with severe developmental abnormalities. These data suggest that in addition to its critical role in seed oil formation, DGAT1 enzyme may also be important for normal seed development in B. napus, although the underlying mechanism(s) remain to be determined.

An N-terminal fragment of mouse DGAT1 binds different acyl-CoAs with varying affinity

A histidine-tagged recombinant N-terminal fragment of type-1 mouse liver diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), MmDGAT1(1-95)His6, was expressed in Escherichia coli, and used to investigate possible acyl-CoA-binding properties. Analysis of the purified fragment by MALDI-TOF mass spectrometry revealed a polypeptide with molecular mass of about 11 kDa which was consistent with the calculated molecular mass based on the deduced amino acid sequence. Lipidex-1000 binding assays indicated that MmDGAT1(1-95)His(6) interacted with long chain fatty acyl-CoAs similar to observations on DGAT1 from oilseed rape (Brassica napus). Binding, as a function of acyl-CoA concentration, differed for palmitoyl (16:0), stearoyl (18:0), and erucoyl (cisDelta(13)22:1)-CoA. Binding of stearoyl- or erucoyl-CoA to MmDGAT1(1-95)His(6) as a function of acyl-CoA concentration, however, was sigmoid and displayed positive cooperativity suggesting that MmDGAT1 may be subject to allosteric modulation by acyl-CoAs. An intra-polypeptide segment within the N-terminal region of MmDGAT1 contained remnants of an acyl-CoA-binding signature initially identified in plant DGAT1. The acyl-CoA-binding site in mammalian DGAT1 could represent a potential target for therapeutic interventions for disorders such as type-2 diabetes and obesity.

Role of cysteine residues in thiol modification of acyl-CoA:diacylglycerol acyltransferase 2 from yeast.

Acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20, DGAT or DAGAT), which catalyzes the final step in triacylglycerol biosynthesis, has at least two discrete family members (DGAT1 and DGAT2) with different physiological roles. Here we report a systematic study of the local functional and structural role of seven cysteine residues present in DGAT2 from Saccharomyces cerevisiae (ScDGAT2, also known as Dga1p) using chemical modification in combination with site-directed mutagenesis. We demonstrate that although DGAT2 was susceptible to various thiol-modifying reagents, none of the cysteines were directly involved in the catalytic activity. Analysis of the accessibility of the sulfhydryl groups revealed that cysteines are also not involved in formation of intramolecular disulfide linkages. Inhibition of DGAT activity with thiol-specific reagents was localized to cysteine 314, which was found to be in the proximity of a highly conserved motif of DGAT2. Our work indicates that although this cysteine does not play a role in enzymatic catalysis, it may reside in a crucial position that is near a possible active site of DGAT2 or related to proper folding of the protein.