scholarly journals Type II Diacylglycerol Acyltransferase from Claviceps purpurea with Ricinoleic Acid, a Hydroxyl Fatty Acid of Industrial Importance, as Preferred Substrate

2009 ◽  
Vol 76 (4) ◽  
pp. 1135-1142 ◽  
Author(s):  
Ioannis Mavraganis ◽  
Dauenpen Meesapyodsuk ◽  
Patricia Vrinten ◽  
Mark Smith ◽  
Xiao Qiu
Keyword(s):  
Fatty Acid ◽  
Ricinoleic Acid ◽  
Biochemical Characterization ◽  
Diacylglycerol Acyltransferase ◽  
Acyl Donor ◽  
Claviceps Purpurea ◽  
Type Ii ◽  
Acyl Acceptor

ABSTRACT Claviceps purpurea, the fungal pathogen that causes the cereal disease ergot, produces glycerides that contain high levels of ricinoleic acid [(R)-12-hydroxyoctadec-cis-9-enoic acid] in its sclerotia. Recently, a fatty acid hydroxylase (C. purpurea FAH [CpFAH]) involved in the biosynthesis of ricinoleic acid was identified from this fungus (D. Meesapyodsuk and X. Qiu, Plant Physiol. 147:1325-1333, 2008). Here, we describe the cloning and biochemical characterization of a C. purpurea type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of ricinoleic acid into triglycerides. The CpDGAT2 gene was cloned by degenerate RT-PCR (reverse transcription-PCR). The expression of this gene restored the in vivo synthesis of triacylglycerol (TAG) in the quadruple mutant strain Saccharomyces cerevisiae H1246, in which all four TAG biosynthesis genes (DGA1, LRO1, ARE1, and ARE2) are disrupted. In vitro enzymatic assays using microsomal preparations from the transformed yeast strain indicated that CpDGAT2 prefers ricinoleic acid as an acyl donor over linoleic acid, oleic acid, or linolenic acid, and it prefers 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as an acyl acceptor. The coexpression of CpFAH with CpDGAT2 in yeast resulted in an increased accumulation of ricinoleic acid compared to the coexpression of CpFAH with the native yeast DGAT2 (S. cerevisiae DGA1 [ScDGA1]) or the expression of CpFAH alone. Northern blot analysis indicated that CpFAH is expressed solely in sclerotium cells, with no transcripts of this gene being detected in mycelium or conidial cells. CpDGAT2 was more widely expressed among the cell types examined, although expression was low in conidiospores. The high expression of CpDGAT2 and CpFAH in sclerotium cells, where high levels of ricinoleate glycerides accumulate, provided further evidence supporting the roles of CpDGAT2 and CpFAH as key enzymes for the synthesis and assembly of ricinoleic acid in C. purpurea.

Substrate preferences of long-chain acyl-CoA synthetase and diacylglycerol acyltransferase contribute to enrichment of flax seed oil with α-linolenic acid

2018 ◽  
Vol 475 (8) ◽  
pp. 1473-1489 ◽  
Author(s):  
Yang Xu ◽  
Roman Holic ◽  
Darren Li ◽  
Xue Pan ◽  
Elzbieta Mietkiewska ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Linolenic Acid ◽  
Seed Oil ◽  
Diacylglycerol Acyltransferase ◽  
Acyl Donor ◽  
Flax Seed ◽  
Chain Acyl ◽  
Long Chain

Seed oil from flax (Linum usitatissimum) is enriched in α-linolenic acid (ALA; 18:3Δ9cis,12cis,15cis), but the biochemical processes underlying the enrichment of flax seed oil with this polyunsaturated fatty acid are not fully elucidated. Here, a potential process involving the catalytic actions of long-chain acyl-CoA synthetase (LACS) and diacylglycerol acyltransferase (DGAT) is proposed for ALA enrichment in triacylglycerol (TAG). LACS catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which in turn may serve as an acyl-donor in the DGAT-catalyzed reaction leading to TAG. To test this hypothesis, flax LACS and DGAT cDNAs were functionally expressed in Saccharomyces cerevisiae strains to probe their possible involvement in the enrichment of TAG with ALA. Among the identified flax LACSs, LuLACS8A exhibited significantly enhanced specificity for ALA over oleic acid (18:1Δ9cis) or linoleic acid (18:2Δ9cis,12cis). Enhanced α-linolenoyl-CoA specificity was also observed in the enzymatic assay of flax DGAT2 (LuDGAT2-3), which displayed ∼20 times increased preference toward α-linolenoyl-CoA over oleoyl-CoA. Moreover, when LuLACS8A and LuDGAT2-3 were co-expressed in yeast, both in vitro and in vivo experiments indicated that the ALA-containing TAG enrichment process was operative between LuLACS8A- and LuDGAT2-3-catalyzed reactions. Overall, the results support the hypothesis that the cooperation between the reactions catalyzed by LACS8 and DGAT2 may represent a route to enrich ALA production in the flax seed oil.

FATTY ACID ETHYL ESTER FORMATION BY PLASMA IN VITRO

1966 ◽  
Vol 44 (2) ◽  
pp. 219-227 ◽  
Author(s):  
W. H. Newsome ◽  
J. B. M. Rattray
Keyword(s):  
Fatty Acid ◽  
Acid Ethyl Ester ◽  
Fatty Acyl ◽  
Ethyl Esters ◽  
Acyl Donor ◽  
Significant Activity ◽  
Fatty Acid Substrate ◽  
Ester Formation

The capacity of rat plasma to form ethyl esters when incubated with ethanol and fatty acid was examined. The process was found to be enzymatic and to involve primarily a direct esterification of fatty acid as opposed to a transesterification requiring a fatty acyl donor. Maximal esterification of oleic acid occurred at pH 6.0 but significant activity existed at physiological pH to indicate a capacity of the plasma to utilize ethanol and fatty acid in concentrations that might be expected in vivo. Both normal and post-heparin plasma were found to esterify endogenous free fatty acid. A major factor affecting the esterification process was the availability of fatty acid substrate and the governing role of plasma albumin in this respect is discussed.

scholarly journals The Role of Type II Fatty Acid Synthesis Enzymes FabZ, ODSCI, and ODSCII in the Pathogenesis of Toxoplasma gondii Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiao-Pei Xu ◽  
Hany M. Elsheikha ◽  
Wen-Ge Liu ◽  
Zhi-Wei Zhang ◽  
Li-Xiu Sun ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Toxoplasma Gondii ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Plaque Assay ◽  
Acid Synthesis ◽  
Host Cells ◽  
Type Ii

Toxoplasma gondii is an obligate intracellular protozoan parasite, which has a worldwide distribution and can infect a large number of warm-blooded animals and humans. T. gondii must colonize and proliferate inside the host cells in order to maintain its own survival by securing essential nutrients for the development of the newly generated tachyzoites. The type II fatty acid biosynthesis pathway (FASII) in the apicoplast is essential for the growth and survival of T. gondii. We investigated whether deletion of genes in the FASII pathway influences the in vitro growth and in vivo virulence of T. gondii. We focused on beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ) and oxidoreductase, short chain dehydrogenase/reductase family proteins ODSCI and ODSCII. We constructed T. gondii strains deficient in FabZ, ODSCI, and ODSCII using CRISPR-Cas9 gene editing technology. The results of immunofluorescence assay, plaque assay, proliferation assay and egress assay showed that in RHΔFabZ strain the apicoplast was partly lost and the growth ability of the parasite in vitro was significantly inhibited, while for RHΔODSCI and RHΔODSCII mutant strains no similar changes were detected. RHΔFabZ exhibited reduced virulence for mice compared with RHΔODSCI and RHΔODSCII, as shown by the improved survival rate. Deletion of FabZ in the PRU strain significantly decreased the brain cyst burden in mice compared with PRUΔODSCI and PRUΔODSCII. Collectively, these findings suggest that FabZ contributes to the growth and virulence of T. gondii, while ODSCI and ODSCII do not contribute to these traits.

Triacylglycerol synthesis and diacylglycerol acyltransferase activity during skeletal myogenesis

1990 ◽  
Vol 68 (12) ◽  
pp. 1393-1401 ◽  
Author(s):  
Victor S. Sauro ◽  
Kenneth P. Strickland
Keyword(s):  
Fatty Acid ◽  
Unsaturated Fatty Acids ◽  
Diacylglycerol Acyltransferase ◽  
Skeletal Myogenesis ◽  
Acyltransferase Activity ◽  
Triacylglycerol Synthesis ◽  
Tag Accumulation ◽  
Long Chain

The role that diacylglycerol acyltransferase (DAGAT) may play in the switch in lipid metabolism from predominantly triacylglycerol- and phospholipid-synthesizing myoblasts to predominantly phospholipid-synthesizing myotubes has been studied during L6 skeletal myogenesis. Fatty acid induced triacylglycerol (TAG) accumulation in vivo was found to be optimal with long-chain, unsaturated fatty acids. The fatty acid induced TAG accumulation was significantly greater in myoblasts than that in myotubes. DAGAT activity in vitro was found to be associated with the particulate (membrane) fraction only. The inhibition by many thiol-specific reagents (N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, 5,5′-dithiobis(2-nitrobenzoic acid)) suggest that a thiol group is at or near the active site. In general, optimal DAGAT activity in vitro was observed when long-chain unsaturated acyl-CoAs and diacylglycerols (DAGs) containing long acyl chains were used as substrates for in vitro TAG synthesis (although 1,2-didecanoin was also very effective). DAGAT activity (expressed relative to DNA) was shown to decline over twofold during skeletal myogenesis when measured in the absence of exogenous DAG. However, in the presence of exogenous (1 mM) DAG, there was no significant change in DAGAT activity, suggesting that the levels of this enzyme are not altered during skeletal myogenesis. These results indicate that endogenous DAG levels are limiting TAG synthesis in L6 myotubes. However, DAG content of myotubes was significantly greater than that of myoblasts, suggesting that there may be an increased competition for DAG (perhaps owing to enhanced phospholipid synthesis) during skeletal myogenesis. The combined effects of decreased synthesis and increased degradation (reported earlier) of TAG may account for the decrease in endogenous TAG contents observed during skeletal myogenesis.Key words: diacylglycerol acyltransferase, TAG synthesis, skeletal myogenesis.

The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

1993 ◽  
Vol 51 ◽  
pp. 74-75
Author(s):  
Jason R. Swedlow ◽  
Neil Osheroff ◽  
Tim Karr ◽  
John W. Sedat ◽  
David A. Agard
Keyword(s):  
Topoisomerase Ii ◽  
Biochemical Characterization ◽  
Developmental Cycle ◽  
Dna Topoisomerase Ii ◽  
Chromosomal Distribution ◽  
Dna Topoisomerase ◽  
Ideal System ◽  
Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

scholarly journals Phenotypic Analysis of Mice Lacking the Tmprss2-Encoded Protease

2006 ◽  
Vol 26 (3) ◽  
pp. 965-975 ◽  
Author(s):  
Tom S. Kim ◽  
Cynthia Heinlein ◽  
Robert C. Hackman ◽  
Peter S. Nelson
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Biological Activities ◽  
Type Ii ◽  
Phenotypic Analysis ◽  
Normal Prostate ◽  
Prostate Hyperplasia ◽  
Transmembrane Serine Protease

ABSTRACT Tmprss2 encodes an androgen-regulated type II transmembrane serine protease (TTSP) expressed highly in normal prostate epithelium and has been implicated in prostate carcinogenesis. Although in vitro studies suggest protease-activated receptor 2 may be a substrate for TMPRSS2, the in vivo biological activities of TMPRSS2 remain unknown. We generated Tmprss2 −/− mice by disrupting the serine protease domain through homologous recombination. Compared to wild-type littermates, Tmprss2 −/− mice developed normally, survived to adulthood with no differences in protein levels of prostatic secretions, and exhibited no discernible abnormalities in organ histology or function. Loss of TMPRSS2 serine protease activity did not influence fertility, reduce survival, result in prostate hyperplasia or carcinoma, or alter prostatic luminal epithelial cell regrowth following castration and androgen replacement. Lack of an observable phenotype in Tmprss2 −/− mice was not due to transcriptional compensation by closely related Tmprss2 homologs. We conclude that the lack of a discernible phenotype in Tmprss2 −/− mice suggests functional redundancy involving one or more of the type II transmembrane serine protease family members or other serine proteases. Alternatively, TMPRSS2 may contribute a specialized but nonvital function that is apparent only in the context of stress, disease, or other systemic perturbation.

scholarly journals Synthesis, Quantification, and Characterization of Fatty Acid Amides from In Vitro and In Vivo Sources

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2543
Author(s):  
Ruidong Ni ◽  
Suzeeta Bhandari ◽  
Perry R. Mitchell ◽  
Gabriela Suarez ◽  
Neel B. Patel ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Fatty Acid ◽  
Apis Mellifera ◽  
Chemical Synthesis ◽  
Acid Amide ◽  
Fatty Acid Amides ◽  
Fatty Acid Amide

Fatty acid amides are a diverse family of underappreciated, biologically occurring lipids. Herein, the methods for the chemical synthesis and subsequent characterization of specific members of the fatty acid amide family are described. The synthetically prepared fatty acid amides and those obtained commercially are used as standards for the characterization and quantification of the fatty acid amides produced by biological systems, a fatty acid amidome. The fatty acid amidomes from mouse N18TG2 cells, sheep choroid plexus cells, Drosophila melanogaster, Bombyx mori, Apis mellifera, and Tribolium castaneum are presented.

scholarly journals Effect of a Rumen-Protected Microencapsulated Supplement from Linseed Oil on the Growth Performance, Meat Quality, and Fatty Acid Composition in Korean Native Steers

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1253
Author(s):  
Chae-Hyung Sun ◽  
Jae-Sung Lee ◽  
Jalil Ghassemi Nejad ◽  
Won-Seob Kim ◽  
Hong-Gu Lee
Keyword(s):  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Growth Performance ◽  
Meat Quality ◽  
Acid Composition ◽  
Linseed Oil ◽  
Control Group ◽  
Ruminal Fluid

We evaluated the effects of a rumen-protected microencapsulated supplement from linseed oil (MO) on ruminal fluid, growth performance, meat quality, and fatty acid composition in Korean native steers. In an in vitro experiment, ruminal fluid was taken from two fistulated Holstein dairy cows. Different levels of MO (0%, 1%, 2%, 3%, and 4%) were added to the diet. In an in vivo experiment, eight steers (average body weight = 597.1 ± 50.26 kg; average age = 23.8 ± 0.12 months) were assigned to two dietary groups, no MO (control) and MO (3% MO supplementation on a DM basis), for 186 days. The in vitro study revealed that 3% MO is an optimal dose, as there were decreases in the neutral detergent fiber and acid detergent fiber digestibility at 48 h (p < 0.05). The in vivo study showed increases in the feed efficiency and average daily gain in the 3% MO group compared to the control group on days 1 to 90 (p < 0.05). Regarding meat quality, the shear force produced by the longissimus thoracis muscle in steers from the 3% MO group was lower than that produced by the control group (p < 0.05). Interestingly, in terms of the fatty acid profile, higher concentrations of C22:6n3 were demonstrated in the subcutaneous fat and higher concentrations of C18:3n3, C20:3n3, and C20:5n3 were found in the intramuscular fat from steers fed with 3% MO (p < 0.05). Our results indicate that supplementation with 3% MO supplements improves the growth performance and meat quality modulated by the omega-3 fatty acid content of meat in Korean native steers.

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