Showing papers on "Acyltransferase published in 2013"
TL;DR: The central importance of the de novo and recycling pathways to PtdOH in cell physiology suggest that these enzymes are suitable targets for the development of antibacterial therapeutics in Gram-positive pathogens.
150 citations
TL;DR: This review provides a detailed survey of the wide spectrum of bacterial acyltransferases and compares different enzyme families in regard to their catalytic mechanisms.
Abstract: SUMMARY Long-chain-length hydrophobic acyl residues play a vital role in a multitude of essential biological structures and processes. They build the inner hydrophobic layers of biological membranes, are converted to intracellular storage compounds, and are used to modify protein properties or function as membrane anchors, to name only a few functions. Acyl thioesters are transferred by acyltransferases or transacylases to a variety of different substrates or are polymerized to lipophilic storage compounds. Lipases represent another important enzyme class dealing with fatty acyl chains; however, they cannot be regarded as acyltransferases in the strict sense. This review provides a detailed survey of the wide spectrum of bacterial acyltransferases and compares different enzyme families in regard to their catalytic mechanisms. On the basis of their studied or assumed mechanisms, most of the acyl-transferring enzymes can be divided into two groups. The majority of enzymes discussed in this review employ a conserved acyltransferase motif with an invariant histidine residue, followed by an acidic amino acid residue, and their catalytic mechanism is characterized by a noncovalent transition state. In contrast to that, lipases rely on completely different mechanism which employs a catalytic triad and functions via the formation of covalent intermediates. This is, for example, similar to the mechanism which has been suggested for polyester synthases. Consequently, although the presented enzyme types neither share homology nor have a common three-dimensional structure, and although they deal with greatly varying molecule structures, this variety is not reflected in their mechanisms, all of which rely on a catalytically active histidine residue.
126 citations
TL;DR: Time-course transcriptome analyses were carried out during the lipid biosynthesis and accumulation processes of the model green alga Chlamydomonas reinhardtii using the Illumina RNA-seq platform, indicating that over 2500 genes are upregulated or during lipid accumulation compared to log phase growth.
108 citations
TL;DR: It is demonstrated that disruption of PDAT1 in the tgd1-1 mutant background causes serious growth retardation, gametophytic defects and premature cell death in developing leaves, and a critical role for PDat1 in mediating TAG synthesis and thereby protecting against FFA-induced cellDeath in fast-growing tissues of plants is revealed.
Abstract: Summary
Phospholipid:diacylglycerol acyltransferase (PDAT) and diacylglycerol:acyl CoA acyltransferase play overlapping roles in triacylglycerol (TAG) assembly in Arabidopsis, and are essential for seed and pollen development, but the functional importance of PDAT in vegetative tissues remains largely unknown. Taking advantage of the Arabidopsis tgd1–1 mutant that accumulates oil in vegetative tissues, we demonstrate here that PDAT1 is crucial for TAG biosynthesis in growing tissues. We show that disruption of PDAT1 in the tgd1–1 mutant background causes serious growth retardation, gametophytic defects and premature cell death in developing leaves. Lipid analysis data indicated that knockout of PDAT1 results in increases in the levels of free fatty acids (FFAs) and diacylglycerol. In vivo 14C-acetate labeling experiments showed that, compared with wild-type, tgd1–1 exhibits a 3.8-fold higher rate of fatty acid synthesis (FAS), which is unaffected by disruption or over-expression of PDAT1, indicating a lack of feedback regulation of FAS in tgd1–1. We also show that detached leaves of both pdat1–2 and tgd1–1 pdat1–2 display increased sensitivity to FFA but not to diacylglycerol. Taken together, our results reveal a critical role for PDAT1 in mediating TAG synthesis and thereby protecting against FFA-induced cell death in fast-growing tissues of plants.
99 citations
TL;DR: GPAT1, but not GPAT4, metabolizes FA synthesized de novo from [14C]acetate and diverts FA away from mitochondrial oxidation and to divert them away from oxidation.
79 citations
TL;DR: Results allow us to conclude that AtDAGT2 is a functional acyl‐CoA:diacylglycerol acyltransferase enzyme that can catalyse substantial increase in TAG synthesis.
72 citations
TL;DR: Functional analysis of enzyme activity in vivo demonstrated that expression of PtDGAT2B can increase the proportion of unsaturated C16 and C18 fatty acids in yeast TAG, suggesting that TAG synthesis and lipid body formation could be completely restored in a Saccharomyces cerevisiae TAG‐deficient quadruple mutant by expressing PtD GAT2 B.
72 citations
TL;DR: Dioscin shows excellent protective effect against ethanol-induced liver injury through amelioratinganol-induced oxidative stress, mitochondrial function, inflammatory cytokine production, apoptosis and liver steatosis, which should be developed as a new drug for the treatment of ethanol- induced liver injury in the future.
Abstract: Our previous studies have shown that dioscin has protective effect against liver injury. However, the action of the compound against ethanol-induced liver injury is still unknown. In the present paper, ethanol-induced acute and chronic liver damage rat models were used, and the results showed that dioscin significantly alleviated liver steatosis, reduced the levels of alanine aminotransferase, aspartate aminotransferase, total triglyceride (TG), total cholesterol and malondialdehyde, and increased the levels of high-density lipoprotein, superoxide dismutase, glutathione and glutathione peroxidase. Transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays showed that dioscin prevented mitochondrial ultrastructural alterations and apoptosis caused by ethanol. In addition, dioscin significantly inhibited ethanol-induced cytochrome P450 2E1 activation, down-regulated the levels of mitogen-activated protein kinases phosphorylation, inhibited the expressions of nuclear factor kappa B, glucose regulated protein 78, activating transcription factor 6 and alpha subunit of translation initiation factor 2 to attenuate oxidative damage, decreased the expressions of tumor necrosis factor alpha and interleukin-6, and down-regulated the expressions of apoptosis-related proteins including p53, caspase-3, caspase-9, poly (ADP-ribose)-polymerase and cytokeratin-18. Further investigation indicated that dioscin markedly increased the expressions of peroxisome proliferators-activated receptor α and its target genes including medium-chain acyl-CoA dehydrogenase, carnitine palmitoyl-CoA transferase I and acyl-CoA oxidase to advance fatty acid β-oxidation, up-regulated the expressions of acyl-CoA synthetase long-chain family member 1, acyl-CoA synthetase long-chain family member 5, alpha-aminoadipic semialdehyde dehydrogenase and acyl-CoA dehydrogenase to promote fatty acid metabolism, and down-regulated the expressions of glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase 1 and diacylglycerol acyltransferase 2 to accelerate TG synthesis. However, dioscin had no effects on the expressions of sterol regulatory element-binding protein-1c, fatty acid synthase, acetyl-CoA carboxylase 1 and stearoyl-CoA desaturase 1 associated with fatty acid synthesis. In conclusion, dioscin shows excellent protective effect against ethanol-induced liver injury through ameliorating ethanol-induced oxidative stress, mitochondrial function, inflammatory cytokine production, apoptosis and liver steatosis, which should be developed as a new drug for the treatment of ethanol-induced liver injury in the future.
70 citations
TL;DR: It is shown that whereas the specificity for the electrophilic malonyl or methylmalonyl component is primarily expressed in the first half ofreaction (formation of the acyl-enzyme intermediate), the second half-reaction shows comparable specificity forthe acyl carrier protein that carries the nucleophilic pantetheine arm.
Abstract: Acyltransferase (AT) domains of modular polyketide synthases exercise tight control over the choice of α-carboxyacyl-CoA substrates, but the mechanistic basis for this specificity is unknown. We show that whereas the specificity for the electrophilic malonyl or methylmalonyl component is primarily expressed in the first half-reaction (formation of the acyl–enzyme intermediate), the second half-reaction shows comparable specificity for the acyl carrier protein that carries the nucleophilic pantetheine arm. We also show that currently used approaches for engineering AT domain specificity work mainly by degrading specificity for the natural substrate rather than by enhancing specificity for alternative substrates.
60 citations
TL;DR: Insect bioassay results showed that mrGAT is required for full fungal virulence by aiding fungal penetration of host cuticles and data from this study suggest that filamentous fungi such as M. robertsii can serve as a good model to elucidate the role of the glycerol phosphate pathway in fungal physiology, particularly to determine the mechanistic connection of GPAT to LD formation.
Abstract: Enzymes involved in the triacylglycerol (TAG) biosynthesis have been well studied in the model organisms of yeasts and animals Among these, the isoforms of glycerol-3-phosphate acyltransferase (GPAT) redundantly catalyze the first and rate-limiting step in glycerolipid synthesis Here, we report the functions of mrGAT, a GPAT ortholog, in an insect-pathogenic fungus, Metarhizium robertsii Unlike in yeasts and animals, a single copy of the mrGAT gene is present in the fungal genome and the gene deletion mutant is viable Compared to the wild type and the gene-rescued mutant, the ΔmrGAT mutant demonstrated reduced abilities to produce conidia and synthesize TAG, glycerol, and total lipids More importantly, we found that mrGAT is localized to the endoplasmic reticulum and directly linked to the formation of lipid droplets (LDs) in fungal cells Insect bioassay results showed that mrGAT is required for full fungal virulence by aiding fungal penetration of host cuticles Data from this study not only advance our understanding of GPAT functions in fungi but also suggest that filamentous fungi such as M robertsii can serve as a good model to elucidate the role of the glycerol phosphate pathway in fungal physiology, particularly to determine the mechanistic connection of GPAT to LD formation
55 citations
TL;DR: To assess the abilities of commercial wine lactic acid bacteria to synthesize potentially flavour active fatty acid ethyl esters and determine mechanisms involved in their production, a Nanofiltration Facility is established at the University of Wisconsin-Madison.
Abstract: Aims
To assess the abilities of commercial wine lactic acid bacteria (LAB) to synthesize potentially flavour active fatty acid ethyl esters and determine mechanisms involved in their production.
Methods and Results
Oenococcus oeni AWRI B551 produced significant levels of ethyl hexanoate and ethyl octanoate following growth in an ethanolic test medium, and ester formation generally increased with increasing pH (4·5 > 3·5), anaerobiosis and precursor supplementation. Cell-free extracts of commercial O. oeni strains and Lactobacillus plantarum AWRI B740 were also tested for ester-synthesizing capabilities in a phosphate buffer via: (i) acyl coenzyme A: alcohol acyltransferase (AcoAAAT) activity and (ii) reverse esterase activity. For both ester-synthesizing activities, strain-dependent variation was observed, with AcoAAAT activity generally greater than reverse esterase. Reverse esterase in O. oeni AWRI B551 also esterified 1-propanol to produce propyl octanoate, and deuterated substrates ([2H6]ethanol and [2H15]octanoic acid) to produce the fully deuterated ester, [2H5]ethyl [2H15]octanoate.
Conclusions
Wine LAB exhibit ethyl ester-synthesizing capability and possess two different ester-synthesizing activities, one of which is associated with an acyl coenzyme A: alcohol acyltransferase.
Significance and Impact of the Study
This study demonstrates that wine LAB exhibit enzyme activities that can augment the ethyl ester content of wine. This knowledge will facilitate greater control over the impacts of malolactic fermentation on the fruity sensory properties and quality of wine.
TL;DR: The findings suggest that miR-9 might be an important regulator in cellular cholesterol homeostasis and decrease the formation of foam cells in vivo by reducing ACAT1 proteins.
Abstract: MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by targeting mRNAs and control a wide range of biological functions. Recent studies have indicated that miRNAs can regulate lipid and cholesterol metabolism in mammals. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a key enzyme in cellular cholesterol metabolism. The accumulated cholesteryl esters are mainly synthesized by ACAT1 during the formation of foam cell, a hallmark of early atherosclerotic lesions. Here, we revealed that miR-9 could target the 3'-untranslated region of human ACAT1 mRNA, specifically reduce human ACAT1 or reporter firefly luciferase (Fluc) proteins but not their mRNAs in different human cell lines, and functionally decrease the formation of foam cells from THP-1-derived macrophages. Our findings suggest that miR-9 might be an important regulator in cellular cholesterol homeostasis and decrease the formation of foam cells in vivo by reducing ACAT1 proteins.
TL;DR: A dominant mutant, dwarf and round leaf-1 (drl1-D), which exhibits weak BR-deficient or BR-insensitive mutant phenotypes, including short and round leaves, prolonged senescence, dwarfed shape, and altered expression levels of the BR-responsive genes is identified.
TL;DR: It is proposed that the activity of Tgl3p is restricted to lipid droplets, whereas the endoplasmic reticulum may serve as a parking lot for this enzyme, as shown by enzymatic analysis and lipid profiling.
TL;DR: The identification and functional characterization of diacylglycerol acyl transferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in Rhodotorula glutinis, and a soluble acyl-CoA-dependent DAG acyltransferase from R. glutinis that belongs to the DGAT3 class of enzymes are described.
Abstract: 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.
TL;DR: This study determined the structures of FadD10 in both the apo-form and the complexed form with dodecanoyl-AMP, where it is seen for the first time an adenylate-forming enzyme that does not adopt a closed conformation for catalysis, which contradicts the existing model of FAALs that rely on an insertion motif for the acyltransferase specificity.
TL;DR: BAT1 (BR-related acyltransferase 1), a gene encoding a putative acyl transferase, was found to be involved in vascular bundle development in a full-length cDNA over-expressor (FOX) screen, suggesting that BAT1 is involved in BR homeostasis, probably by conversion of brassinolide intermediates into acylated BR conjugates.
Abstract: Summary
Brassinosteroids (BRs) are essential for various aspects of plant development. Cellular BR homeostasis is critical for proper growth and development of plants; however, its regulatory mechanism remains largely unknown. BAT1 (BR-related acyltransferase 1), a gene encoding a putative acyltransferase, was found to be involved in vascular bundle development in a full-length cDNA over-expressor (FOX) screen. Over-expression of BAT1 resulted in typical BR-deficient phenotypes, which were rescued by exogenously applied castasterone and brassinolide. Analyses of BR profiles demonstrated that BAT1 alters levels of several brassinolide biosynthetic intermediates, including 6-deoxotyphasterol, typhasterol and 6-deoxocastasterone. BAT1 is mainly localized in the endoplasmic reticulum. BAT1 is highly expressed in young tissues and vascular bundles, and its expression is induced by auxin. These data suggest that BAT1 is involved in BR homeostasis, probably by conversion of brassinolide intermediates into acylated BR conjugates.
TL;DR: The X-ray structure of LnmK represents the first AT enzyme that employs a Tyr as an active site residue and the first member of the family of double-hot-dog fold enzymes that displays an AT activity known to date.
Abstract: The β-branched C3 unit in leinamycin biosynthesis is installed by a set of four proteins, LnmFKLM. In vitro biochemical investigation confirmed that LnmK is a bifunctional acyltransferase/decarboxylase (AT/DC) that catalyzes first self-acylation using methylmalonyl-CoA as a substrate and subsequently transacylation of the methylmalonyl group to the phosphopantetheinyl group of the LnmL acyl carrier protein [Liu, T., Huang, Y., and Shen, B. (2009) J. Am. Chem. Soc. 131, 6900-6901]. LnmK shows no sequence homology to proteins of known function, representing a new family of AT/DC enzymes. Here we report the X-ray structure of LnmK. LnmK is homodimer with each of the monomers adopting a double-hot-dog fold. Cocrystallization of LnmK with methylmalonyl-CoA revealed an active site tunnel terminated by residues from the dimer interface. In contrast to canonical AT and ketosynthase enzymes that employ Ser or Cys as an active site residue, none of these residues are found in the vicinity of the LnmK active site. Instead, three tyrosines were identified, one of which, Tyr62, was established, by site-directed mutagenesis, to be the most likely active site residue for the AT activity of LnmK. LnmK represents the first AT enzyme that employs a Tyr as an active site residue and the first member of the family of double-hot-dog fold enzymes that displays an AT activity known to date. The LnmK structure sets the stage for probing of the DC activity of LnmK through site-directed mutagenesis. These findings highlight natural product biosynthetic machinery as a rich source of novel enzyme activities, mechanisms, and structures.
TL;DR: Results suggest that PLA/AT-1 is at least partly responsible for the generation of NAPE in mammalian cells.
TL;DR: The in vivo and in vitro characterization of AntB, a dedicated acyltransferase encoded in the antimycin biosynthetic gene cluster, which catalyzes the C-8 acyloxy formation is reported.
TL;DR: Lipoproteins are triacylated in slow-growing mycobacteria after exploiting the vaccine strain Mycobacterium bovis BCG as a model organism to investigate lipoprotein modifications inSlow-growingMycob bacteria.
Abstract: Background
Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid.
TL;DR: An enzymatic production method for synthesizing L-alanyl-L- glutamine (Ala-Gln) from L-alanine methyl ester hydrochloride and L-glutamines was developed in this study and the molar yield was 67% against both AlaOMe and Gln.
Abstract: An enzymatic production method for synthesizing L-alanyl-L-glutamine (Ala-Gln) from L-alanine methyl ester hydrochloride (AlaOMe) and L-glutamine (Gln) was developed in this study. The cultivation conditions for an Escherichia coli strain overexpressing α-amino acid ester acyltransferase from Sphingobacterium siyangensis AJ 2458 (SAET) and reaction conditions for Ala-Gln production were optimized. A high cell density culture broth prepared by fed-batch cultivation showed 440 units/mL of Ala-Gln-producing activity. In addition, an Ala-Gln-producing reaction using intact E. coli cells overexpressing SAET under optimum conditions was conducted. A total Ala-Gln yield of 69.7 g/L was produced in 40 min. The molar yield was 67% against both AlaOMe and Gln.
TL;DR: Overexpression of Sct1p increased the extent of remodeling of PE-derived PC, providing a novel tool to perform mechanistic studies on PC acyl chain exchange and indicating that PC serves as a buffer for excess cellular C16:0.
TL;DR: This is the first characterized algal DGAT3 gene, giving further evidence to the occurrence of a DG AT3-mediated TAG biosynthesis pathway.
Abstract: In this study, a cDNA encoding a novel acyl-CoA:diacylglycerol acyltransferase (DGAT)-like protein is identified and isolated from the diatom microalga Phaeodactylum tricornutum (PtDGAT3). Analysis of the sequence reveals that ptDGAT3 cDNA encodes a protein of 504 amino acids with a molecular mass of 64.5 KDa. The putative ptDGAT3 protein has two catalytic domains: a wax ester synthase-like acyl-CoA acyltransferase domain and a bacteria-specific acyltransferase domain, which shows higher similarity to the DGAT3 of Acinetobacter calcoaceticus than reported DGAT1 or DGAT2 from high plants or algae. Its activity was confirmed by heterologous expression of PtDGAT3 in a neutral lipid-deficient quadruple mutant yeast Saccharomyces cerevisiae H1246. The recombinant yeast restored the formation of a lipid body and displayed a preference to the incorporation of unsaturated C-18 fatty acids into triacyglycerol (TAG). This is the first characterized algal DGAT3 gene, giving further evidence to the occurrence of a DGAT3-mediated TAG biosynthesis pathway.
TL;DR: The results suggested that, in addition to the role in breaking down storage lipids, yeast peroxisomes also contribute to lipid synthesis by recycling acyl‐CoAs when a fatty acyl assembly system is available to capture and utilize the fatty acy pools generated via β‐oxidation.
Abstract: Glycerolipids of the marine diatom Thalassiosira pseudonana are enriched particularly with eicosapentaenoic acid (EPA), and also with an appreciable level of docosahexaenoic acid (DHA). The present study describes the functional characterization of a type 2 diacylglycerol acyltransferase (DGAT2, EC 2.3.1.20) from T. pseudonana, designated TpDGAT2, which catalyzes the final step of triacylglycerol (TAG) synthesis. Heterologous expression of this gene restored TAG formation in a yeast mutant devoid of TAG biosynthesis. TpDGAT2 was also shown to exert a large impact on the fatty acid profile of TAG. Its expression caused a 10-12% increase of 18:1 and a concomitant decrease of 16:0 relative to that of AtDGAT1(Arabidopsis thaliana). Furthermore, in the presence of the very-long-chain polyunsaturated fatty acids (VLCPUFA) EPA and DHA, TAG formed by TpDGAT2 displayed three- to six-fold increases in the percentage of VLCPUFA relative to that of AtDGAT1 even though TpDGAT2 conferred much lower TAG-synthetic activities than Arabidopsis DGAT1. Strikingly, when fed DHA, the yeast mutant expressing TpDGAT2 incorporated high levels of EPA and DHA isomers derived from DHA β-oxidation. In contrast, no such phenomenon occurred in the cells expressing AtDGAT1. These results suggested that, in addition to the role in breaking down storage lipids, yeast peroxisomes also contribute to lipid synthesis by recycling acyl-CoAs when a fatty acyl assembly system is available to capture and utilize the fatty acyl pools generated via β-oxidation. Our study hence illustrated a case where the efficiency and substrate preference of an acyltransferase can elicit far reaching metabolic adjustments that affect TAG composition.
TL;DR: The identified small subunit of serine palmitoyltransferase a (ssSPTa) as an LPIAT1‐interacting protein suggests that ssSPTa plays a role in fatty acid remodeling of PI, probably by facilitating the MAM localization of LPIat1.
Abstract: Lysophosphatidylinositol acyltransferase 1 (LPIAT1), also known as MBOAT7, is a phospholipid acyltransferase that selectively incorporates arachidonic acid (AA) into the sn-2 position of phosphatidylinositol (PI). We previously demonstrated that LPIAT1 regulates AA content in PI and plays a crucial role in brain development in mice. However, how LPIAT1 is regulated and which proteins function cooperatively with LPIAT1 are unknown. In this study, using a split-ubiquitin membrane yeast two-hybrid system, we identified the small subunit of serine palmitoyltransferase a (ssSPTa) as an LPIAT1-interacting protein. ssSPTa co-immunoprecipitated and colocalized with LPIAT1 in cultured mammalian cells. Knockdown of ssSPTa decreased the LPIAT1-dependent incorporation of exogenous AA into PI but did not affect the in vitro enzyme activity of LPIAT1 in the microsomal fraction. Interestingly, knockdown of ssSPTa decreased the protein level of LPIAT1 in the crude mitochondrial fraction but not in total homogenate or the microsomal fraction. LPIAT1 was localized to the mitochondria-associated membrane (MAM), where AA-selective acyl-CoA synthetase is enriched. These results suggest that ssSPTa plays a role in fatty acid remodeling of PI, probably by facilitating the MAM localization of LPIAT1.
TL;DR: Cl cloning and characterization of GPAT gene from Lepidium latifolium suggests presence of a single copy of the gene in the genome, and it can be considered a potential candidate gene for genetic engineering of oil yields and abiotic stress management in food as well as fuel crops.
Abstract: Glycerol-3-phosphate acyltransferase (GPAT) catalyzes first and the rate limiting step in glycerolipid synthesis pathway, which in turn contribute to stabilization of plasma membrane structure and oil lipid synthesis in plant cells. Here, we report cloning and characterization of GPAT gene from Lepidium latifolium (LlaGPAT). The cDNA sequence (1,615 bp) of LlaGPAT gene consisted of 1,113 bp ORF encoding a protein of 370 aa residues, with deduced mass of 41.2 kDa and four acyltransferase (AT) motifs having role in catalysis and in glycerol-3-phosphate binding. Southern blot analysis suggested presence of a single copy of the gene in the genome. Tissue specific expression of the gene was seen more abundantly in aerial parts, compared to the roots. Quantitative real-time PCR indicated down-regulation of the gene by cold (4 °C), drought (PEG6000), salt (300 mM NaCl) and ABA (100 μM) treatments. Considering the vitality of the function of encoded enzyme, LlaGPAT can be considered a potential candidate gene for genetic engineering of oil yields and abiotic stress management in food as well as fuel crops.
TL;DR: This work utilizes acyltransferase activity of a new bacterial isolate Bacillus smithii strain IITR6b2 for the synthesis of nicotinic acid hydroxamate (NAH), a heterocyclic class of hydroxamic acid, an important pyridine derivative that has found its role as bioligand, urease inhibitor, antityrosinase, antioxidant, antimetastatic, and vasodilating agents.
Abstract: In this work, acyltransferase activity of a new bacterial isolate Bacillus smithii strain IITR6b2 was utilized for the synthesis of nicotinic acid hydroxamate (NAH), a heterocyclic class of hydroxamic acid. NAH is an important pyridine derivative and has found its role as bioligand, urease inhibitor, antityrosinase, antioxidant, antimetastatic, and vasodilating agents. Amidase having acyltransferase activity with nicotinamide is suitable for nicotinic acid hydroxamate production. However, amidase can also simultaneously hydrolyze nicotinamide and nicotinic acid hydroxamate to nicotinic acid. Nicotinic acid is an undesirable by-product and thus any biocatalytic process involving amidase for nicotinic acid hydroxamate production needs to have high ratios of acyltransferase to amide hydrolase and acyltransferase to nicotinic acid hydroxamate hydrolase activity. Isolate Bacillus smithii strain IITR6b2 was found to have 28- and 12.3-fold higher acyltransferase to amide and hydroxamic acid hydrolase activities, respectively. This higher ratio resulted in a limited undesirable by-product, nicotinic acid (NA) synthesis. The optimal substrate/co-substrate ratio, pH, temperature, incubation time, and resting cells concentration were 200/250 mM, 7, 30 °C, 40 min, and 0.7 mgDCW ml−1, respectively, and 94.5 % molar conversion of nicotinamide to nicotinic acid hydroxamate was achieved under these reaction conditions. To avoid substrate inhibition effect, a fed-batch process based on the optimized parameters with two feedings of substrates (200/200 mM) at 40-min intervals was developed and a molar conversion yield of 89.4 % with the productivity of 52.9 g h−1 gDCW−1 was achieved at laboratory scale. Finally, 6.4 g of powder containing 58.5 % (w/w) nicotinic acid hydroxamate was recovered after lyophilization and further purification resulted in 95 % pure product.
TL;DR: Sequence and phylogeny analyses indicated that genes in all four groups of the MBOAT gene models encode enzymes with acyltransferases, which suggested their important roles in adaptation of plants in lands.
Abstract: Membrane bound O-acyl transferase (MBOAT) family is composed of gene members encoding a variety of acyltransferase enzymes, which play important roles in plant acyl lipid metabolism. Here, we present the first genome-enabled identification and analysis of MBOAT gene models in plants. In total, we identified 136 plant MBOAT sequences from 14 plant species with complete genomes. Phylogenetic relationship analyses suggested the plant MBOAT gene models fell into four major groups, two of which likely encode enzymes of diacylglycerol acyltransferase 1 (DGAT1) and lysophospholipid acyltransferase (LPLAT), respectively, with one–three copies of paralogs present in each of the most plant species. A group of gene sequences, which are homologous to Saccharomyces cerevisiae glycerol uptake proteins (GUP), was identified in plants; copy numbers were conserved, with only one copy represented in each of the most plant species; analyses showed that residues essential for acyltransferases were more prone to be conserved than vertebrate orthologs. Among four groups, one was inferred to emerge in land plants and experience a rapid expansion in genomes of angiosperms, which suggested their important roles in adaptation of plants in lands. Sequence and phylogeny analyses indicated that genes in all four groups encode enzymes with acyltransferases. Comprehensive sequence identification of MBOAT family members and investigation into classification provide a complete picture of the MBOAT gene family in plants, and could shed light into enzymatic functions of different MBOAT genes in plants.
TL;DR: This chapter describes a convenient LCAT assay based on the use of an apoA-I mimetic peptide, which is easily made with the peptide and can be stored by deep freezing without significant loss of activity.
Abstract: Lecithin-cholesterol acyltransferase (LCAT) is the major enzyme responsible for the esterification of free cholesterol on plasma lipoproteins, which is a key step in the reverse cholesterol transport pathway. The measurement of plasma LCAT activity not only is important in the diagnosis of patients with genetic or acquired LCAT deficiency but is also valuable in calculating cardiovascular risk, as well as in research studies of lipoprotein metabolism. In this chapter, we describe a convenient LCAT assay based on the use of an apoA-I mimetic peptide. The proteoliposome substrate used in this assay for LCAT is easily made with the peptide and can be stored by deep freezing without significant loss of activity.