A look into DGAT1 through the EM lenses.

With the advent of modern detectors and robust structure solution pipeline, cryogenic electron microscopy has recently proved to be game changer in structural biology. Membrane proteins are challenging targets for structural biologists. This minireview focuses a membrane embedded triglyceride synthesizing machine, DGAT1. Decades of research had built the foundational knowledge on this enzyme's activity. However, recently solved cryo-EM structures of this enzyme, in apo and bound form, has provided critical mechanistic insights. The flipping of the catalytic histidine is critical of enzyme catalysis. The structures explain why the enzyme has preference to long fatty acyl chains over the short forms.

Acyl-CoA:diacylglycerol acyltransferase: Properties, physiological roles, metabolic engineering and intentional control.

Acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the last reaction in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG). DGAT activity resides mainly in DGAT1 and DGAT2 in eukaryotes and bifunctional wax ester synthase-diacylglycerol acyltransferase (WSD) in bacteria, which are all membrane-bound proteins but exhibit no sequence homology to each other. Recent studies also identified other DGAT enzymes such as the soluble DGAT3 and diacylglycerol acetyltransferase (EaDAcT), as well as enzymes with DGAT activities including defective in cuticular ridges (DCR) and steryl and phytyl ester synthases (PESs). This review comprehensively discusses research advances on DGATs in prokaryotes and eukaryotes with a focus on their biochemical properties, physiological roles, and biotechnological and therapeutic applications. The review begins with a discussion of DGAT assay methods, followed by a systematic discussion of TAG biosynthesis and the properties and physiological role of DGATs. Thereafter, the review discusses the three-dimensional structure and insights into mechanism of action of human DGAT1, and the modeled DGAT1 from Brassica napus. The review then examines metabolic engineering strategies involving manipulation of DGAT, followed by a discussion of its therapeutic applications. DGAT in relation to improvement of traits of farmed animals is also discussed along with DGATs in various other eukaryotic organisms.

Highly accurate protein structure prediction for the human proteome.

Protein structures can provide invaluable information, both for reasoning about biological processes and for enabling interventions such as structure-based drug development or targeted mutagenesis. After decades of effort, 17% of the total residues in human protein sequences are covered by an experimentally determined structure1. Here we markedly expand the structural coverage of the proteome by applying the state-of-the-art machine learning method, AlphaFold2, at a scale that covers almost the entire human proteome (98.5% of human proteins). The resulting dataset covers 58% of residues with a confident prediction, of which a subset (36% of all residues) have very high confidence. We introduce several metrics developed by building on the AlphaFold model and use them to interpret the dataset, identifying strong multi-domain predictions as well as regions that are likely to be disordered. Finally, we provide some case studies to illustrate how high-quality predictions could be used to generate biological hypotheses. We are making our predictions freely available to the community and anticipate that routine large-scale and high-accuracy structure prediction will become an important tool that will allow new questions to be addressed from a structural perspective.

Discovery of novel DGAT1 inhibitors by combination of machine learning methods, pharmacophore model and 3D-QSAR model.

DGAT1 plays a crucial controlling role in triglyceride biosynthetic pathways, which makes it an attractive therapeutic target for obesity. Thus, development of DGAT1 inhibitors with novel chemical scaffolds is desired and important in the drug discovery. In this investigation, the multistep virtual screening methods, including machine learning methods and common feature pharmacophore model, were developed and used to identify novel DGAT1 inhibitors from BioDiversity database with 30,000 compounds. 531 compounds were predicted as DGAT1 inhibitors by combination of machine learning methods comprising of SVM, NB and RP models. Then, 12 agents were filtered from 531 compounds by using the common feature pharmacophore model. The 3D chemical structures of the 12 hits coordinated with surface charges and isosurface have been carefully analyzed by the established 3D-QSAR model. Finally, 8 compounds with desired properties were retained from the final hits and have been assigned to another research group to complete the follow-up compound synthesis and biologic evaluation.

Functional characterization of two type-1 diacylglycerol acyltransferase (DGAT1) genes from rice (Oryza sativa) embryo restoring the triacylglycerol accumulation in yeast.

KEY MESSAGE: Two OsDGAT1 genes showed the ability to restore TAG and LB synthesis in yeast H1246. Alterations in the N-terminal region of OsDGAT1-1 gene revealed its regulatory role in gene function. Accumulation of triacylglycerol (TAG) or oil in vegetative tissues has emerged as a promising approach to meet the global needs of food, feed, and fuel. Rice (Oryza sativa) has been recognized as an important cereal crop containing nutritional rice bran oil with high economic value for renewable energy production. To identify the key component involved in storage lipid biosynthesis, two type-1 diacylglycerol acyltransferases (DGAT1) from rice were characterized for its in vivo function in the H1246 (dga1, lro1, are1 and are2) yeast quadruple mutant. The ectopic expression of rice DGAT1 (designated as OsDGAT1-1 and OsDGAT1-2) genes restored the capability of TAG synthesis and lipid body (LB) formation in H1246. OsDGAT1-1 showed nearly equal substrate preferences to C16:0-CoA and 18:1-CoA whereas OsDGAT1-2 displayed substrate selectivity for C16:0-CoA over 18:1-CoA, indicating that these enzymes have contrasting substrate specificities. In parallel, we have identified the intrinsically disordered region (IDR) at the N-terminal domains of OsDGAT1 proteins. The regulatory role of the N-terminal domain was dissected. Single point mutations at the phosphorylation sites and truncations of the N-terminal region highlighted reduced lipid accumulation capabilities among different OsDGAT1-1 variants.

Characterization of diacylglycerol acyltransferase 2 from Idesia polycarpa and function analysis.

Idesia polycarpa is an oil-producing tree native to China and Northeast Asia. The fruits of I. polycarpa which are named oil grape are unique in that they contain large amounts saturated and unsaturated lipids. Diacylglycerol acyltransferase 2 (DGAT2) is a key enzyme catalyzing the final step of triacylglyceride (TAG) synthesis. However, expression and bioinformatics of DGAT2 in I. polycarpa are still blank. In order to further understand the lipogenesis of oil grape, we contrasted seven various growth periods fruits from seed formation to seed maturation. Lipid accumulation rates and final lipid content were significantly different among the different periods. We cloned and characterized the DGAT2 gene from fruits of I. polycarpa. A partial fragment of 239 bp of IpDGAT2 was amplified by PCR. We cloned the open-reading frame (ORF) of IpDGAT2 by RACE technique. The ORF of IpDGAT2 contains 984 bp and encodes 327 amino acids. The qPCR analysis manifested that IpDGAT2 was expressed in all oil grape growing periods and expression was highest on September 20 (seed maturation). In I. polycarpa fruits the expression of IpDGAT2 was positively correlated with the lipid accumulation rates. Rhodotorula glutinis expression analysis showed that IpDGAT2 have a diacylglycerol acyltransferase bio-functional. Heterologous expression of the 35S::IpDGAT2 in Arabidopsis thaliana confirmed that the isolated IpDGAT2 could catalyze lipid synthesis. The lipid content increased by 40 % in transgenic plants relative to the control. which suggests that high lipid content fruits can be created by the overexpression of IpDGAT2 in I. polycarpa.

Structure and mechanism of human diacylglycerol O-acyltransferase 1.

Diacylglycerol O-acyltransferase 1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in humans1. DGAT1 belongs to the membrane-bound O-acyltransferase (MBOAT) superfamily, members of which are found in all kingdoms of life and are involved in the acylation of lipids and proteins2,3. How human DGAT1 and other mammalian members of the MBOAT family recognize their substrates and catalyse their reactions is unknown. The absence of three-dimensional structures also hampers rational targeting of DGAT1 for therapeutic purposes. Here we present the cryo-electron microscopy structure of human DGAT1 in complex with an oleoyl-CoA substrate. Each DGAT1 protomer has nine transmembrane helices, eight of which form a conserved structural fold that we name the MBOAT fold. The MBOAT fold in DGAT1 forms a hollow chamber in the membrane that encloses highly conserved catalytic residues. The chamber has separate entrances for each of the two substrates, fatty acyl-CoA and diacylglycerol. DGAT1 can exist as either a homodimer or a homotetramer and the two forms have similar enzymatic activity. The N terminus of DGAT1 interacts with the neighbouring protomer and these interactions are required for enzymatic activity.

Structure and catalytic mechanism of a human triacylglycerol-synthesis enzyme.

Triacylglycerols store metabolic energy in organisms and have industrial uses as foods and fuels. Excessive accumulation of triacylglycerols in humans causes obesity and is associated with metabolic diseases1. Triacylglycerol synthesis is catalysed by acyl-CoA diacylglycerol acyltransferase (DGAT) enzymes2-4, the structures and catalytic mechanisms of which remain unknown. Here we determined the structure of dimeric human DGAT1, a member of the membrane-bound O-acyltransferase (MBOAT) family, by cryo-electron microscopy at approximately 3.0 Å resolution. DGAT1 forms a homodimer through N-terminal segments and a hydrophobic interface, with putative active sites within the membrane region. A structure obtained with oleoyl-CoA substrate resolved at approximately 3.2 Å shows that the CoA moiety binds DGAT1 on the cytosolic side and the acyl group lies deep within a hydrophobic channel, positioning the acyl-CoA thioester bond near an invariant catalytic histidine residue. The reaction centre is located inside a large cavity, which opens laterally to the membrane bilayer, providing lipid access to the active site. A lipid-like density-possibly representing an acyl-acceptor molecule-is located within the reaction centre, orthogonal to acyl-CoA. Insights provided by the DGAT1 structures, together with mutagenesis and functional studies, provide the basis for a model of the catalysis of triacylglycerol synthesis by DGAT.

Characterization of a Type-2 Diacylglycerol Acyltransferase from Haematococcus pluvialis Reveals Possible Allostery of the Recombinant Enzyme.

Haematococcus pluvialis is a green microalga used in the algal biotechnology industry that can accumulate considerable amounts of storage triacylglycerol (TAG) and astaxanthin, which is a high-value carotenoid with strong antioxidant activity, under stress conditions. Diacylglycerol acyltransferase (DGAT) catalyzes the last step of the acyl-CoA-dependent TAG biosynthesis and appears to represent a bottleneck in algal TAG formation. In this study, putative H. pluvialis DGAT2 cDNA (HpDGAT2A, B, D and E) were identified from a transcriptome database and were subjected to sequence-based in silico analyses. The coding sequences of HpDGAT2B, D, and E were then isolated and characterized through heterologous expression in a TAG-deficient Saccharomyces cerevisiae strain H1246. The expression of HpDGAT2D allowed the recovery of TAG biosynthesis in this yeast mutant, and further in vitro enzymatic assays confirmed that the recombinant HpDGAT2D possessed strong DGAT activity. Interestingly, the recombinant HpDGAT2D displayed sigmoidal kinetics in response to increasing acyl-CoA concentrations, which has not been reported in plant or algal DGAT2 in previous studies.

Exploring novel single nucleotide polymorphisms and haplotypes of the diacylglycerol O-acyltransferase 1 (DGAT1) gene and their effects on protein structure in Iranian buffalo.

BACKGROUND: Diacylglycerol O-acyltransferase 1 (DGAT1) plays a key role in the synthesis of triglycerides. Recent studies have shown that a transition mutation resulting in substitutions of guanine by adenine in the DGAT1 gene in cattle has considerable effects on milk yield and composition. Currently, there is no systematic research reporting on the utilization of this gene segment in Iranian buffalo (Bubalus bubalis). OBJECTIVE: In this study, the genetic differentiation of three indigenous Iranian buffalo populations was investigated in the region spanning exon 3 to exon 17 of the DGAT1 gene. METHODS: A total of 200 buffaloes were genotyped, all the samples were sequenced directly in both directions with forward and reverse sequencing primers. RESULTS: Sequence analysis showed novel SNPs compared to the reference GenBank sequence (DQ886485) at nucleotide positions g.6097A>G, g.7036C>T, g.7338G>A, g.7710C>T, g.8087C>T, g.8259G>A, g.8275G>A, g.8367C>T, and g.8426C>T. No polymorphisms were found within exon 8. Therefore, the K232A position was thought to be a conserved and fixed region for high milk fat content (K allele) in Bos indicus and all buffalo breeds. Comparison with Indian buffalo revealed three exonic SNPs, one of which was nonsynonymous. A unique 22 bp insertion was observed in intron 10 of DGAT1. Linkage disequilibrium analysis allowed the identification of nine haplotypes among the sampled animals. To our knowledge, this is the first report of sequencing analysis of the DGAT1 gene in Iranian buffalo. CONCLUSION: Our results suggest that genetic diversity exists and could be useful in examining the association between the DGAT1 gene and milk production traits in buffalo.

Directed evolution of a bacterial WS/DGAT acyltransferase: improving tDGAT from Thermomonospora curvata.

Some bacteria belonging to the actinobacteria and proteobacteria groups can accumulate neutral lipids expressing enzymes of the wax ester synthase/acyl coenzyme A: diacylglycerol acyltransferase (WS/DGAT) family. tDGAT is a WS/DGAT-like enzyme from Thermomonospora curvata able to produce TAGs and WEs when heterologously expressed in Escherichia coli. In this study, a protocol for the directed evolution of bacterial lipid-producing enzymes based on fluorimetry is developed and tested. tDGAT has been successfully evolved towards the improvement of TAG production with an up to 2.5 times increase in TAG accumulation. Mutants with no ability to produce TAGs but able to accumulate waxes were also selected during the screening. The localization of the mutations that enhance TAG production in the outer surface of tDGAT points out possible new mechanisms that contribute to the activity of this family of enzymes. This Nile red-based high throughput screening provides an evolution platform for other WS/DGAT-like enzymes.

The Amphibian Diacylglycerol O-acyltransferase 2 (DGAT2): a 'paleo-protein' with Conserved Function but Unique Folding.

Amphibians are, currently, considered the first vertebrates that had performed the aquatic to terrestrial transition during evolution; therefore, water balance and dehydration control were prerequisites for such environment conquering. Among anurans, Phyllomedusa is a well-studied genus, due to its peptide-rich skin secretion. Here, we have analyzed the skin secretion of Phyllomedusa distincta targeting the proteins present in the skin secretion. The major soluble protein was chromatographically isolated and utilized to immunize rabbits. Through proteomics approaches, we were able to identify such protein as being the diacylglycerol O-acyltransferase 2 (DGAT2), a crucial enzyme involved in lipid synthesis and in the skin water balance. Immunohistochemistry assays revealed the protein tissular distribution for different animal species, belonging to different branches of the phylogenetic tree. Specifically, there was positivity to the anti-DGAT2 on Amphibians' skin, and no antibody recognition on fish and mammals' skins. The DGAT2 multiple sequence alignment reveals some degree of conservation throughout the genera; however, there is a different cysteine pattern among them. Molecular modeling analyses corroborate that the different cysteine pattern leads to distinct 3D structures, explaining the different antibody recognition. Moreover, the protein phylogenetic analyses place the Xenopus DGAT2 (the available amphibian representative) next to the Coelacanthus enzyme, which have led the authors to term this a 'paleo-protein'. DGAT2 would be, therefore, an ancient protein, crucial to the terrestrial environment conquest, with a unique folding-as indicated by the molecular models and immunohistochemistry analyses-a consequence of the different cysteine pattern but with conserved biological function.

Characterization of Type-2 Diacylglycerol Acyltransferases in the Green Microalga Chromochloris zofingiensis.

Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step of the acyl-CoA-dependent TAG biosynthesis and thus is a key target for manipulating oil production in microalgae. The microalga Chromochloris zofingiensis can accumulate substantial amounts of triacylglycerol (TAG) and represents a promising source of algal lipids. In this study, C. zofingiensis DGAT2s (CzDGAT2s) were characterized with in silico, in vivo (yeast), and in vitro assays. Putative CzDGAT2s were identified, and their functional motifs and evolutionary relationship with other DGAT2s were analyzed. When CzDGAT2s were individually expressed in a TAG-deficient Saccharomyces cerevisiae strain, only CzDGAT2C could restore the TAG biosynthesis. Further in vitro assays indicated that CzDGAT2C displayed typical DGAT activity, which was fitted to the Michaelis-Menten equation, and N- and C-terminals were important for the enzyme activity. In addition, membrane yeast two-hybrid assay revealed a possible DGAT2 activity modulation via the formation of homodimer/heterodimer among different DGAT2 isoforms.

Newly Identified Essential Amino Acids Affecting Chlorella ellipsoidea DGAT1 Function Revealed by Site-Directed Mutagenesis.

Diacylglycerol acyltransferase (DGAT) is a rate-limiting enzyme in the synthesis of triacylglycerol (TAG), the most important form of energy storage in plants. Some residues have previously been proven to be crucial for DGAT1 activity. In this study, we used site-directed mutagenesis of the CeDGAT1 gene from Chlorella ellipsoidea to alter 16 amino acids to investigate effects on DGAT1 function. Of the 16 residues (L482R, E542R, Y553A, G577R, R579D, Y582R, R596D, H603D, H609D, A624R, F629R, S632A, W650R, A651R, Q658H, and P660R), we newly identified 5 (L482, R579, H603, A651, and P660) as being essential for DGAT1 function and 7 (E542, G577, R596, H609, A624, S632, and Q658) that significantly affect DGAT1 function to different degrees, as revealed by heterologous expression of the mutants in yeast strain INVSc1. Importantly, compared with CeDGAT1, expression of the mutant CeDGAT1Y553A significantly increased the total fatty acid and TAG contents of INVSc1. Comparison among CeDGAT1Y553A, GmDGAT1Y341A, AtDGAT1Y364A, BnDGAT1Y347A, and BoDGAT1Y352A, in which tyrosine at the position corresponding to the 553rd residue in CeDGAT1 is changed into alanine, indicated that the impact of changing Y to A at position 553 is specific for CeDGAT1. Overall, the results provide novel insight into the structure and function of DGAT1, as well as a mutant gene with high potential for lipid improvement in microalgae and plants.

Arabidopsis thaliana DGAT3 is a [2Fe-2S] protein involved in TAG biosynthesis.

Acyl-CoA:diacylglycerol acyltransferases 3 (DGAT3) are described as plant cytosolic enzymes synthesizing triacylglycerol. Their protein sequences exhibit a thioredoxin-like ferredoxin domain typical of a class of ferredoxins harboring a [2Fe-2S] cluster. The Arabidopsis thaliana DGAT3 (AtDGAT3; At1g48300) protein is detected in germinating seeds. The recombinant purified protein produced from Escherichia coli, although very unstable, exhibits DGAT activity in vitro. A shorter protein version devoid of its N-terminal putative chloroplast transit peptide, Δ46AtDGAT3, was more stable in vitro, allowing biochemical and spectroscopic characterization. The results obtained demonstrate the presence of a [2Fe-2S] cluster in the protein. To date, AtDGAT3 is the first metalloprotein described as a DGAT.

Intrinsic disorder in the regulatory N-terminal domain of diacylglycerol acyltransferase 1 from Brassica napus.

Proteins with multifunctional regulatory domains often demonstrate structural plasticity or protein disorder, allowing the binding of multiple regulatory factors and post-translational modifications. While the importance of protein disorder is clear, it also poses a challenge for in vitro characterization. Here, we report protein intrinsic disorder in a plant molecular system, which despite its prevalence is less studied. We present a detailed biophysical characterization of the entire cytoplasmic N-terminal domain of Brassica napus diacylglycerol acyltransferase, (DGAT1), which includes an inhibitory module and allosteric binding sites. Our results demonstrate that the monomeric N-terminal domain can be stabilized for biophysical characterization and is largely intrinsically disordered in solution. This domain interacts with allosteric modulators of DGAT1, CoA and oleoyl-CoA, at micromolar concentrations. While solution scattering studies indicate conformational heterogeneity in the N-terminal domain of DGAT1, there is a small gain of secondary structure induced by ligand binding.

Role of DGAT enzymes in triacylglycerol metabolism.

The esterification of a fatty acyl moiety to diacylglycerol to form triacylglycerol (TAG) is catalysed by two diacylglycerol O-acyltransferases (DGATs) encoded by genes belonging to two distinct gene families. The enzymes are referred to as DGAT1 and DGAT2 in order of their identification. Both proteins are transmembrane proteins localized in the endoplasmic reticulum. Their membrane topologies are however significantly different. This difference is hypothesized to give the two isozymes different abilities to interact with other proteins and organelles and access to different pools of fatty acids, thereby creating a distinction between the enzymes in terms of their role and contribution to lipid metabolism. DGAT1 is proposed to have dual topology contributing to TAG synthesis on both sides of the ER membrane and esterifying only the pre-formed fatty acids. There is evidence to suggest that DGAT2 translocates to the lipid droplet (LD), associates with other proteins, and synthesizes cytosolic and luminal apolipoprotein B associated LD-TAG from both endogenous and exogenous fatty acids. The aim of this review is to differentiate between the two DGAT enzymes by comparing the genes that encode them, their proposed topologies, the proteins they interact with, and their roles in lipid metabolism.

Genome-wide characterization and expression profiling of diacylglycerol acyltransferase genes from maize.

Diacylglycerol acyltransferase (DGAT) catalyzes the only rate-limiting step in the pathway of plant oil (TAG) biosynthesis and is involved in plant development. In this study, five DGAT family members were identified from maize genome database. Phylogenetic analysis classified the ZmDGATs into type-I, II, and III clusters. Conserved functional domain analysis revealed that the proteins encoded by ZmDGAT1 contained conserved MBOAT domains, while two ZmDGAT2-encoding proteins harbored LPLAT domains. qRT-PCR analysis showed that ZmDGAT genes exhibited very high relative expression in developing seeds, especially at the early stage of seed development. Under various abiotic stress conditions, differential responses of ZmDGAT genes were observed. An overall significant induction of ZmDGAT genes under cold stress in leaves and a quick and strong response to osmotic stresses in roots were highlighted. This study provides useful information for understanding the roles of DGATs in oil accumulation and stress responses in higher plants.

Diacylglycerol acyltransferase-2 contains a c-terminal sequence that interacts with lipid droplets.

Diacylglycerol acyltranferase-2 (DGAT2) is a resident protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerol. When lipid droplet formation is stimulated by incubating cells with fatty acids, DGAT2 becomes concentrated around the surface of cytosolic lipid droplets. Using confocal microscopy and directed mutagenesis, we have identified a 17-amino acid sequence in the C-terminal region of DGAT2 that is necessary and sufficient for targeting DGAT2 to lipid droplets. When this region was deleted, DGAT2 remained in the ER and did not target to lipid droplets. Fusing this sequence to mCherry directed the fluorescent reporter to lipid droplets. Similarly, when the corresponding region of monoacylglycerol acyltransferase-2 (MGAT2) was replaced with this sequence, MGAT2 was also targeted to lipid droplets. Lastly, we demonstrated that DGAT2 in ER membranes is continuous with lipid droplets. We propose a new model whereby DGAT2 remains in the ER during lipid droplet formation via it's transmembrane domains and interacts with nascent lipid droplets via its C-terminal lipid droplet interacting domain as they expand.

Identification and Characterization of Diacylglycerol Acyltransferase from Oleaginous Fungus Mucor circinelloides.

Acyl-CoA:diacylglycerol acyltransferase (DGAT) is a pivotal regulator of triacylglycerol (TAG) synthesis. The oleaginous fungus Mucor circinelloides has four putative DGATs: McDGAT1A, McDGAT1B, McDGAT2A, and McDGAT2B, classified into the DGAT1 and DGAT2 subfamilies, respectively. To identify and characterize DGATs in M. circinelloides, these four genes were expressed in Saccharomyces cerevisiae H1246 (TAG-deficient quadruple mutant), individually. TAG biosynthesis was restored only by the expression of McDGAT2B, and TAG content was significantly higher in the mutants with McDGAT2B expression than in a S. cerevisiae mutant with endogenous DGA1 expression. McDGAT2B prefers saturated fatty acids to monounsaturated fatty acids and has an obvious preference for C18:3 (ω-6) according to the results of substrate preference experiments. Furthermore, only the mRNA expression pattern of McDGAT2B correlated with TAG biosynthesis during a fermentation process. Our experiments strongly indicate that McDGAT2B is crucial for TAG accumulation, suggesting that it may be an essential target for metabolic engineering aimed at increasing lipid content of M. circinelloides.