scholarly journals Functional Analysis of an Acyltransferase-Like Domain from Polyunsaturated Fatty Acid Synthase in Thraustochytrium

2021 ◽  
Vol 9 (3) ◽  
pp. 626
Author(s):  
Carla Almendáriz-Palacios ◽  
Dauenpen Meesapyodsuk ◽  
Xiao Qiu
Keyword(s):  
Fatty Acids ◽  
Polyunsaturated Fatty Acids ◽  
Free Fatty Acids ◽  
Fatty Acid Synthase ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Complementation Test ◽  
Temperature Sensitive ◽  
Synthetase Activity

Biosynthesis of very long chain polyunsaturated fatty acids (VLCPUFA) such as docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) and docosapentaenoic acid (DPA, 22:5-4,7,10,13,16) in protist Thraustochytrium is catalyzed by a polyunsaturated fatty acids (PUFA) synthase comprising three large subunits, each with multiple catalytic domains. This study used complementation test, in vitro assays, and functional expression to characterize an acyltransferase (AT)-like domain in Subunit-B of a PUFA synthase from Thraustochytrium. Complementation test in Escherichia coli showed that the AT-like domain could not restore the growth phenotype of a temperature-sensitive mutant (∆fabDts) defective in malonyl-CoA:ACP transacylase activity. In vitro assays showed that the AT-like domain possessed thioesterase activity towards a few acyl-CoAs tested where docosahexaenoyl-CoA (DHA-CoA) was the preferred substrate. Expression of this domain in an E. coli mutant (∆fadD) defective in acyl-CoA synthetase activity resulted in the increased accumulation of free fatty acids. Site-directed mutagenesis showed that the substitution of two putative active site residues, serine at 96 (S96) and histidine at 220 (H220), in the AT-like domain significantly reduced its activity towards DHA-CoA and accumulation of free fatty acids in the ∆fadD mutant. These results indicate that the AT-like domain of the PUFA synthase does not function as a malonyl-CoA:ACP transacylase, rather it functions as a thioesterase. It might catalyze the last step of the VLCPUFA biosynthesis by releasing freshly synthesized VLCPUFAs attached to ACP domains of the PUFA synthase in Thraustochytrium.

scholarly journals Diatoms and Plants Acyl-CoA:lysophosphatidylcholine Acyltransferases (LPCATs) Exhibit Diverse Substrate Specificity and Biochemical Properties

2021 ◽  
Vol 22 (16) ◽  
pp. 9056
Author(s):  
Ada Połońska ◽  
Katarzyna Jasieniecka-Gazarkiewicz ◽  
Lingjie You ◽  
Xiahui Hao ◽  
Sylwia Klińska ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Substrate Specificity ◽  
Polyunsaturated Fatty Acids ◽  
Ph Value ◽  
Biochemical Properties ◽  
In Vitro Assays ◽  
Efficient Production ◽  
Genome Database ◽  
Response To Temperature

The search of the Phaeodactylum tricornutum genome database revealed the existence of six genes potentially encoding lysophospholipid acyltransferases. One of these genes, Phatr3_J20460, after introduction to yeast ale1 mutant disrupted in the LPCAT gene, produced a very active acyl-CoA:lysophosphatidylcholine (LPCAT) enzyme. Using in vitro assays applying different radioactive and non-radioactive substrates and microsomal fractions from such yeast, we have characterized the biochemical properties and substrate specificities of this PtLPCAT1. We have found that the substrate specificity of this enzyme indicates that it can completely supply phosphatidylcholine (PC) with all fatty acids connected with a biosynthetic pathway of very long-chain polyunsaturated fatty acids (VLC-PUFAs) used further for the desaturation process. Additionally, we have shown that biochemical properties of the PtLPCAT1 in comparison to plant LPCATs are in some cases similar (such as the dependency of its activity on pH value), differ moderately (such as in response to temperature changes), or express completely different properties (such as in reaction to calcium and magnesium ions or toward some acyl-CoA with 20C polyunsaturated fatty acids). Moreover, the obtained results suggest that cloned “Phatr3_J20460” gene can be useful in oilseeds plant engineering toward efficient production of VLC-PUFA as LPCAT it encodes can (contrary to plant LPCATs) introduce 20:4-CoA (n-3) to PC for further desaturation to 20:5 (EPA, eicosapentaenoic acid).

A temperature-sensitive glycyl-transfer ribonucleic acid synthetase mutant of Escherichia coli

1973 ◽  
Vol 19 (4) ◽  
pp. 421-426 ◽  
Author(s):  
E. R. Roback ◽  
J. D. Friesen ◽  
N. P. Fill
Keyword(s):  
Escherichia Coli ◽  
Ribonucleic Acid ◽  
Bacteriophage T4 ◽  
Temperature Shift ◽  
Transfer Rna ◽  
In Vitro Assays ◽  
Temperature Sensitive ◽  
Heat Sensitivity ◽  
Synthetase Activity

A method of selecting temperature-sensitive mutants of Escherichia coli which makes use of a double temperature shift combined with suicide as a result of incorporated 3H-uridine is described. One mutant selected in this way shows restricted growth at 42 °C resulting from early inhibition of protein and ribonucleic acid (RNA) synthesis. In vitro assays of aminoacyl-transfer RNA synthetase activity indicated a complete absence of active glycyl-transfer RNA synthetase in the mutant. This enzyme activity in the mutant was distinguishable from the wild type by its rapid inactivation at 28 °C in cell-free extracts. Infection of the temperature-sensitive mutant with bacteriophage T4 did not alter the heat-sensitivity of the mutant enzyme. The mutation is 72% cotransducible with the xyl locus.

scholarly journals In vitro intestinal digestion of lipids from the marine diatom Porosira glacialis compared to commercial LC n-3 PUFA products

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252125
Author(s):  
Lars Dalheim ◽  
Jon Brage Svenning ◽  
Ragnar Ludvig Olsen
Keyword(s):  
Fatty Acids ◽  
Polyunsaturated Fatty Acids ◽  
Free Fatty Acids ◽  
Ethyl Ester ◽  
Saturated Fatty Acids ◽  
Marine Diatom ◽  
Cod Liver Oil ◽  
Krill Oil ◽  
Intestinal Digestion

Marine sources of long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) are in high demand for use in health supplements. Mass cultivated marine microalgae is a promising and sustainable source of LC n-3 PUFA, which relieves pressure on natural fish stocks. The lipid class profile from cultivated photosynthetic algae differ from the marine organisms currently used for the production of LC n-3 PUFA. The objective of this study was to compare in vitro intestinal digestion of oil extracted from the cold-adapted marine diatom Porosira glacialis with commercially available LC n-3 PUFA supplements; cod liver oil, krill oil, ethyl ester concentrate, and oil from the copepod Calanus finmarchicus (Calanus® oil). The changes in the free fatty acids and neutral and polar lipids during the enzymatic hydrolysis were characterized by liquid and gas chromatography. In Calanus® oil and the Ethyl ester concentrate, the free fatty acids increased very little (4.0 and 4.6%, respectively) during digestion. In comparison, free fatty acids in Krill oil and P. glacialis oil increased by 14.7 and 17.0%, respectively. Cod liver oil had the highest increase (28.2%) in free fatty acids during the digestion. Monounsaturated and saturated fatty acids were more easily released than polyunsaturated fatty acids in all five oils.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

N-3 Polyunsaturated Fatty Acids Modulate In-Vitro T Cell Function in Type I Diabetic Patients

Lipids ◽  
2008 ◽  
Vol 43 (6) ◽  
pp. 485-497 ◽  
Author(s):  
Sid Ahmed Merzouk ◽  
Meriem Saker ◽  
Karima Briksi Reguig ◽  
Nassima Soulimane ◽  
Hafida Merzouk ◽  
...  
Keyword(s):  
Fatty Acids ◽  
T Cell ◽  
Polyunsaturated Fatty Acids ◽  
Cell Function ◽  
Diabetic Patients ◽  
Type I ◽  
T Cell Function

scholarly journals n‐3 polyunsaturated fatty acids provoke a specific transcriptional profile in rabbit adipose‐derived stem cells in vitro

2019 ◽  
Vol 103 (3) ◽  
pp. 925-934
Author(s):  
Eкaterina Vackova ◽  
Darko Bosnakovski ◽  
Bodil Bjørndal ◽  
Penka Yonkova ◽  
Natalia Grigorova ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Stem Cells ◽  
Polyunsaturated Fatty Acids ◽  
Transcriptional Profile ◽  
Adipose Derived Stem Cells

scholarly journals Quantification of Volatile Aldehydes Deriving from In Vitro Lipid Peroxidation in the Breath of Ventilated Patients

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3089
Author(s):  
Lukas M. Müller-Wirtz ◽  
Daniel Kiefer ◽  
Sven Ruffing ◽  
Timo Brausch ◽  
Tobias Hüppe ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Fatty Acids ◽  
Polyunsaturated Fatty Acids ◽  
Breathing Circuit ◽  
Aliphatic Aldehydes ◽  
Mechanically Ventilated ◽  
Non Invasive ◽  
Plastic Parts ◽  
Ventilated Patients

Exhaled aliphatic aldehydes were proposed as non-invasive biomarkers to detect increased lipid peroxidation in various diseases. As a prelude to clinical application of the multicapillary column–ion mobility spectrometry for the evaluation of aldehyde exhalation, we, therefore: (1) identified the most abundant volatile aliphatic aldehydes originating from in vitro oxidation of various polyunsaturated fatty acids; (2) evaluated emittance of aldehydes from plastic parts of the breathing circuit; (3) conducted a pilot study for in vivo quantification of exhaled aldehydes in mechanically ventilated patients. Pentanal, hexanal, heptanal, and nonanal were quantifiable in the headspace of oxidizing polyunsaturated fatty acids, with pentanal and hexanal predominating. Plastic parts of the breathing circuit emitted hexanal, octanal, nonanal, and decanal, whereby nonanal and decanal were ubiquitous and pentanal or heptanal not being detected. Only pentanal was quantifiable in breath of mechanically ventilated surgical patients with a mean exhaled concentration of 13 ± 5 ppb. An explorative analysis suggested that pentanal exhalation is associated with mechanical power—a measure for the invasiveness of mechanical ventilation. In conclusion, exhaled pentanal is a promising non-invasive biomarker for lipid peroxidation inducing pathologies, and should be evaluated in future clinical studies, particularly for detection of lung injury.

scholarly journals Host/Malassezia Interaction: A Quantitative, Non-Invasive Method Profiling Oxylipin Production Associates Human Skin Eicosanoids with Malassezia

Metabolites ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 700
Author(s):  
Yohannes Abere Ambaw ◽  
Martin P. Pagac ◽  
Antony S. Irudayaswamy ◽  
Manfred Raida ◽  
Anne K. Bendt ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Immune System ◽  
Polyunsaturated Fatty Acids ◽  
Human Skin ◽  
Skin Surface ◽  
Lipid Mediators ◽  
Dependent Manner ◽  
Human Immune System ◽  
Plant Host

Malassezia are common components of human skin, and as the dominant human skin eukaryotic microbe, they take part in complex microbe–host interactions. Other phylogenetically related fungi (including within Ustilagomycotina) communicate with their plant host through bioactive oxygenated polyunsaturated fatty acids, generally known as oxylipins, by regulating the plant immune system to increase their virulence. Oxylipins are similar in structure and function to human eicosanoids, which modulate the human immune system. This study reports the development of a highly sensitive mass-spectrometry-based method to capture and quantify bioactive oxygenated polyunsaturated fatty acids from the human skin surface and in vitro Malassezia cultures. It confirms that Malassezia are capable of synthesizing eicosanoid-like lipid mediators in vitro in a species dependent manner, many of which are found on human skin. This method enables sensitive identification and quantification of bioactive lipid mediators from human skin that may be derived from metabolic pathways shared between skin and its microbial residents. This enables better cross-disciplinary and detailed studies to dissect the interaction between Malassezia and human skin, and to identify potential intervention points to promote or abrogate inflammation and to improve human skin health.

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