scholarly journals Annotation of Genes Involved in Glycerolipid Biosynthesis in Chlamydomonas reinhardtii: Discovery of the Betaine Lipid Synthase BTA1Cr

2005 ◽  
Vol 4 (2) ◽  
pp. 242-252 ◽  
Author(s):  
Wayne R. Riekhof ◽  
Barbara B. Sears ◽  
Christoph Benning
Keyword(s):  
Fatty Acid ◽  
Chlamydomonas Reinhardtii ◽  
Active Sites ◽  
Expressed Sequence Tag ◽  
Membrane Lipid ◽  
Flowering Plants ◽  
Site Directed Mutagenesis ◽  
Genes Encoding ◽  
Betaine Lipid

ABSTRACT Lipid metabolism in flowering plants has been intensely studied, and knowledge regarding the identities of genes encoding components of the major fatty acid and membrane lipid biosynthetic pathways is very extensive. We now present an in silico analysis of fatty acid and glycerolipid metabolism in an algal model, enabled by the recent availability of expressed sequence tag and genomic sequences of Chlamydomonas reinhardtii. Genes encoding proteins involved in membrane biogenesis were predicted on the basis of similarity to proteins with confirmed functions and were organized so as to reconstruct the major pathways of glycerolipid synthesis in Chlamydomonas. This analysis accounts for the majority of genes predicted to encode enzymes involved in anabolic reactions of membrane lipid biosynthesis and compares and contrasts these pathways in Chlamydomonas and flowering plants. As an important result of the bioinformatics analysis, we identified and isolated the C. reinhardtii BTA1 (BTA1Cr ) gene and analyzed the bifunctional protein that it encodes; we predicted this protein to be sufficient for the synthesis of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), a major membrane component in Chlamydomonas. Heterologous expression of BTA1Cr led to DGTS accumulation in Escherichia coli, which normally lacks this lipid, and allowed in vitro analysis of the enzymatic properties of BTA1Cr. In contrast, in the bacterium Rhodobacter sphaeroides, two separate proteins, BtaARs and BtaBRs, are required for the biosynthesis of DGTS. Site-directed mutagenesis of the active sites of the two domains of BTA1Cr allowed us to study their activities separately, demonstrating directly their functional homology to the bacterial orthologs BtaARs and BtaBRs.

scholarly journals Biochemical characteristics of AtFAR2, a fatty acid reductase from Arabidopsis thaliana that reduces fatty acyl-CoA and -ACP substrates into fatty alcohols

2016 ◽  
Vol 63 (3) ◽  
Author(s):  
Thuy T. P. Doan ◽  
Anders S. Carlsson ◽  
Sten Stymne ◽  
Per Hofvander
Keyword(s):  
Arabidopsis Thaliana ◽  
Fatty Acid ◽  
Fatty Acyl ◽  
Fatty Alcohols ◽  
Abnormal Development ◽  
In Planta ◽  
Alcohol Production ◽  
Genes Encoding ◽  
In Vitro Data

Fatty alcohols and derivatives are important for proper deposition of a functional pollen wall. Mutations in specific genes encoding fatty acid reductases (FAR) responsible for fatty alcohol production cause abnormal development of pollen. A disrupted AtFAR2 (MS2) gene in Arabidopsis thaliana results in pollen developing an abnormal exine layer and a reduced fertility phenotype. AtFAR2 has been shown to be targeted to chloroplasts and in a purified form to be specific for acyl-ACP substrates. Here, we present data on the in vitro and in planta characterizations of AtFAR2 from A. thaliana and show that this enzyme has the ability to use both, C16:0-ACP and C16:0-CoA, as substrates to produce C16:0-alcohol. Our results further show that AtFAR2 is highly similar in properties and substrate specificity to AtFAR6 for which in vitro data has been published, and which is also a chloroplast localized enzyme. This suggests that although AtFAR2 is the major enzyme responsible for exine layer functionality, AtFAR6 might provide functional redundancy to AtFAR2.

scholarly journals A Cytochrome b 5 -Containing Plastid-Located Fatty Acid Desaturase from Chlamydomonas reinhardtii

2012 ◽  
Vol 11 (7) ◽  
pp. 856-863 ◽  
Author(s):  
Simone Zäuner ◽  
Wibke Jochum ◽  
Tara Bigorowski ◽  
Christoph Benning
Keyword(s):  
Fatty Acid ◽  
Chlamydomonas Reinhardtii ◽  
Fluorescent Protein ◽  
Acyl Chain ◽  
Transit Peptide ◽  
Primary Electron ◽  
Chloroplast Targeting ◽  
Content Type ◽  
Cell Extracts

ABSTRACT Monogalactosyldiacylglycerol (MGDG) in Chlamydomonas reinhardtii and other green algae contains hexadeca-4,7,10,13-tetraenoic acid (16:4) in the glycerol sn- 2 position. While many genes necessary for the introduction of acyl chain double bonds have been functionally characterized, the Δ4-desaturase remained unknown. Using a phylogenetic comparison, a candidate gene encoding the MGDG-specific Δ4-desaturase from Chlamydomonas (CrΔ4FAD) was identified. CrΔ4FAD shows all characteristic features of a membrane-bound desaturase, including three histidine boxes and a transit peptide for chloroplast targeting. But it also has an N-terminal cytochrome b 5 domain, distinguishing it from other known plastid desaturases. Cytochrome b 5 is the primary electron donor for endoplasmic reticulum (ER) desaturases and is often fused to the desaturase domain in desaturases modifying the carboxyl end of the acyl group. Difference absorbance spectra of the recombinant cytochrome b 5 domain of CrΔ4FAD showed that it is functional in vitro . Green fluorescent protein fusions of CrΔ4FAD localized to the plastid envelope in Chlamydomonas . Interestingly, overproduction of CrΔ4FAD in Chlamydomonas not only increased levels of 16:4 acyl groups in cell extracts but specifically increased the total amount of MGDG. Vice versa, the amount of MGDG was lowered in lines with reduced levels of CrΔ4FAD. These data suggest a link between MGDG molecular species composition and galactolipid abundance in the alga, as well as a specific function for this fatty acid in MGDG.

Acyl carrier protein: structure–function relationships in a conserved multifunctional protein family

2007 ◽  
Vol 85 (6) ◽  
pp. 649-662 ◽  
Author(s):  
David M. Byers ◽  
Huansheng Gong
Keyword(s):  
Fatty Acid ◽  
Active Sites ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Divalent Cations ◽  
Structural Features ◽  
Carrier Protein ◽  
Type I ◽  
Prosthetic Group

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel α helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its “recognition” helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.

scholarly journals Genes encoding proteins regulating fatty acid metabolism and cellular response to lipids are differentially expressed in porcine luminal epithelium during long-term culture

2019 ◽  
Vol 7 (2) ◽  
pp. 58-65
Author(s):  
Magdalena Kulus ◽  
Blanka Borowiec ◽  
Małgorzata Popis ◽  
Piotr Celichowski ◽  
Michal Jeseta ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Epithelial Cells ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
In Vitro Cultivation ◽  
Beta Oxidation ◽  
Physiological Processes ◽  
Genes Encoding ◽  
Fatty Acid Beta Oxidation

AbstractAmong many factors, the epithelium lining the oviductal lumenis very important for the development of the oocyte and its subsequent fertilization. The oviductal epithelium is characterized by the presence of ciliary cells, supporting the movement of cumulus-oocyte complexes towards the uterus. By interacting with the semen, the epithelium of the fallopian tube makes the sperm acquire the ability to fertilize. So far, the exact molecular mechanisms of these changes have not been known. Hence, understanding the metabolism of oviduct epithelial cells and the level of expression of individual groups of genes seems to be a way to deepen the knowledge about the broadly understood reproduction.In our research, we decided to culture oviductal epithelial cells (OECs) in vitro for a long period of time. After 24h, 7, 15 and 30 days, the OECs were harvested, with their RNA isolated. Transcriptomic changes were analyzed using microarrays. The “cellular response to lipid” group was represented by the following genes: MUC1, CYP24A1, KLF4, IL24, SNAI2, CXCL10, PPARD, TNC, ABCA10, while the genes belonging to the “cellular lipid metabolic processes” were: LIPG, ARSK, ACADL, FADS3, P2RX7, ACSS2, PPARD, KITLG, SPTLC3, ERBB3, KLF4, CRABP2. Additionally, PPARD and ACADL were members of the “fatty acid beta-oxidation” ontology group. Our study describes genes that are not directly related to fertility processes. However, significant changes in their expression in in vitro cultured OECs may indicate their usefulness as markers of OECs’ physiological processes.Running title: Fatty acids changes in porcine oviductal epithelial cells in in vitro cultivation

scholarly journals Structural Biology of Mutant IDH2 Inhibition

2014 ◽  
Vol 70 (a1) ◽  
pp. C799-C799
Author(s):  
Byron DeLaBarre ◽  
Fang Wang ◽  
Jeremy Travins ◽  
Stefan Gross ◽  
Erin Artin ◽  
...  
Keyword(s):  
Active Sites ◽  
Tight Binding ◽  
Point Mutations ◽  
Myelogenous Leukemia ◽  
Cancer Therapies ◽  
Idh Mutations ◽  
Genes Encoding ◽  
Potential Applications ◽  
Acute Myelogenous

A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases- 1 and -2 (IDH1, IDH2)[1]. These mutations alter residues in the enzyme active sites and confer a gain-of-function in cancer cells, resulting in the accumulation and secretion of the oncometabolite R (-)-2-hydroxyglutarate (2HG). 2HG is a potent inhibitor of DNA methylating enzymes such as TET2[2]. This suggests a connection between cancer related IDH mutations and aberrant epigenetics. As such, IDH represents an important new druggable target in the pursuit of novel cancer therapies. We have developed a small molecule, AGI-6780, that potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. A crystal structure of AGI-6780 complexed with IDH2/R140Q revealed that the inhibitor binds in an allosteric manner at the dimer interface[3]. While structures of IDH1 and IDH2 were known, this is the first ever structure of an inhibited IDH protein and shows a novel conformation of IDH2. The results of steady-state enzymology analysis were consistent with allostery and slow-tight binding by AGI-6780. Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human acute myelogenous leukemia (AML) cells in vitro. These data provide proof-of- concept that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for cancer.

scholarly journals Biochemical and Physiological Characterization of the Pyruvate Formate-Lyase Pfl1 of Chlamydomonas reinhardtii, a Typically Bacterial Enzyme in a Eukaryotic Alga

2008 ◽  
Vol 7 (3) ◽  
pp. 518-526 ◽  
Author(s):  
Anja Hemschemeier ◽  
Jessica Jacobs ◽  
Thomas Happe
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Anaerobic Metabolism ◽  
Pyruvate Decarboxylase ◽  
Pyruvate Formate Lyase ◽  
Physiological Characterization ◽  
Bacterial Enzyme ◽  
Genes Encoding ◽  
Oxygen Sensitive ◽  
Biochemical Analyses

ABSTRACT The unicellular green alga Chlamydomonas reinhardtii has a special type of anaerobic metabolism that is quite unusual for eukaryotes. It has two oxygen-sensitive [Fe-Fe] hydrogenases (EC 1.12.7.2) that are coupled to photosynthesis and, in addition, a formate- and ethanol-producing fermentative metabolism, which was proposed to be initiated by pyruvate formate-lyase (Pfl; EC 2.3.1.54). Pfl enzymes are commonly found in prokaryotes but only rarely in eukaryotes. Both the hydrogen- and the formate/ethanol-producing pathways are involved in a sustained anaerobic metabolism of the alga, which can be induced by sulfur depletion in illuminated cultures. Before now, the presence of a Pfl protein in C. reinhardtii was predicted from formate secretion and the homology of the deduced protein of the PFL1 gene model to known Pfl enzymes. In this study, we proved the formate-producing activity of the putative Pfl1 enzyme by heterologous expression of the C. reinhardtii PFL1 cDNA in Escherichia coli and subsequent in vitro activity tests of the purified protein. Furthermore, a Pfl-deficient E. coli strain secretes formate when expressing the PFL1 cDNA of C. reinhardtii. We also examined the Pfl1 fermentation pathway of C. reinhardtii under the physiological condition of sulfur depletion. Genetic and biochemical analyses show that sulfur-depleted algae express genes encoding enzymes acting downstream of Pfl1 and also potentially ethanol-producing enzymes, such as pyruvate decarboxylase (EC 4.1.1.1) or pyruvate ferredoxin oxidoreductase (EC 1.2.7.1). The latter enzymes might substitute for Pfl1 activity when Pfl1 is specifically inhibited by hypophosphite.

scholarly journals Probing Direct Interactions between CodY and the oppD Promoter of Lactococcus lactis

2005 ◽  
Vol 187 (2) ◽  
pp. 512-521 ◽  
Author(s):  
Chris D. den Hengst ◽  
Peter Curley ◽  
Rasmus Larsen ◽  
Girbe Buist ◽  
Arjen Nauta ◽  
...  
Keyword(s):  
Amino Acids ◽  
Lactococcus Lactis ◽  
Nitrogen Availability ◽  
Nitrogen Sources ◽  
Dnase I ◽  
Site Directed Mutagenesis ◽  
Upstream Region ◽  
Binding Studies ◽  
Genes Encoding

ABSTRACT CodY of Lactococcus lactis MG1363 is a transcriptional regulator that represses the expression of several genes encoding proteins of the proteolytic system. These genes include pepN, pepC, opp-pepO1, and probably prtPM, pepX, and pepDA2, since the expression of the latter three genes relative to nitrogen availability is similar to that of the former. By means of in vitro DNA binding assays and DNase I footprinting techniques, we demonstrate that L. lactis CodY interacts directly with a region upstream of the promoter of its major target known so far, the opp system. Our results indicate that multiple molecules of CodY interact with this promoter and that the amount of bound CodY molecules is affected by the presence of branched-chain amino acids and not by GTP. Addition of these amino acids strongly affects the extent of the region protected by CodY in DNase I footprints. Random and site-directed mutagenesis of the upstream region of oppD yielded variants that were derepressed in a medium with an excess of nitrogen sources. Binding studies revealed the importance of specific bases in the promoter region required for recognition by CodY.

scholarly journals Activity Mapping the Acyl Carrier Protein - Elongating Ketosynthase Interaction in Fatty Acid Biosynthesis

2020 ◽  
Author(s):  
Jeffrey T. Mindrebo ◽  
Laetitia E. Misson ◽  
Caitlin Johnson ◽  
Joseph P. Noel ◽  
Michael D. Burkart
Keyword(s):  
Fatty Acid ◽  
Active Sites ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Chain Extension ◽  
Carrier Protein ◽  
Type Ii ◽  
Multiple Partner ◽  
Carbon Carbon Bond

ABSTRACTElongating ketosynthases (KSs) catalyze carbon-carbon bond forming reactions during the committed step for each round of chain extension in both fatty acid synthases (FASs) and polyketide synthases (PKSs). A small α-helical acyl carrier protein (ACP) shuttles fatty acyl intermediates between enzyme active sites. To accomplish this task, ACP relies on a series of dynamic interactions with multiple partner enzymes of FAS and associated FAS-dependent pathways. Recent structures of the Escherichia coli FAS ACP, AcpP, in covalent complexes with its two cognate elongating KSs, FabF and FabB, provide high-resolution detail of these interfaces, but a systematic analysis of specific interfacial interactions responsible for stabilizing these complexes has not yet been undertaken. Here, we use site-directed mutagenesis with both in vitro and in vivo activity analyses to quantitatively evaluate these contacting surfaces between AcpP and FabF. We delineate the FabF interface into three interacting regions and demonstrate the effects of point mutants, double mutants, and region delete variants. Results from these analyses reveal a robust and modular FabF interface capable of tolerating seemingly critical interface mutations with only the deletion of entire regions significantly compromising activity. Structure and sequence analysis of FabF orthologs from related type II FAS pathways indicate significant conservation of type II FAS KS interface residues and, overall, support its delineation into interaction regions. These findings strengthen our mechanistic understanding of molecular recognition events between ACPs and FAS enzymes and provide a blueprint for engineering ACP-dependent biosynthetic pathways.

scholarly journals Utility of the Trypanosoma cruzi Sequence Database for Identification of Potential Vaccine Candidates by In Silico and In Vitro Screening

2004 ◽  
Vol 72 (11) ◽  
pp. 6245-6254 ◽  
Author(s):  
Vandanajay Bhatia ◽  
Mala Sinha ◽  
Bruce Luxon ◽  
Nisha Garg
Keyword(s):  
Trypanosoma Cruzi ◽  
Developmental Stages ◽  
Expressed Sequence Tag ◽  
Genomic Sequence ◽  
Genome Mining ◽  
Surface Proteins ◽  
Vaccine Candidates ◽  
Genes Encoding ◽  
Potential Vaccine

ABSTRACT Glycosylphosphatidylinositol (GPI)-anchored proteins are abundantly expressed in the infective and intracellular stages of Trypanosoma cruzi and are recognized as antigenic targets by both the humoral and cellular arms of the immune system. Previously, we demonstrated the efficacy of genes encoding GPI-anchored proteins in eliciting partially protective immunity to T. cruzi infection and disease, suggesting their utility as vaccine candidates. For the identification of additional vaccine targets, in this study we screened the T. cruzi expressed sequence tag (EST) and genomic sequence survey (GSS) databases. By applying a variety of web-based genome-mining tools to the analysis of ∼2,500 sequences, we identified 348 (37.6%) EST and 260 (17.4%) GSS sequences encoding novel parasite-specific proteins. Of these, 19 sequences exhibited the characteristics of secreted and/or membrane-associated GPI proteins. Eight of the selected sequences were amplified to obtain genes TcG1, TcG2, TcG3, TcG4, TcG5, TcG6, TcG7, and TcG8 (TcG1-TcG8) which are expressed in different developmental stages of the parasite and conserved in the genome of a variety of T. cruzi strains. Flow cytometry confirmed the expression of the antigens encoded by the cloned genes as surface proteins in trypomastigote and/or amastigote stages of T. cruzi. When delivered as a DNA vaccine, genes TcG1-TcG6 elicited a parasite-specific antibody response in mice. Except for TcG5, antisera to genes TcG1-TcG6 exhibited trypanolytic activity against the trypomastigote forms of T. cruzi, a property known to correlate with the immune control of T. cruzi. Taken together, our results validate the applicability of bioinformatics in genome mining, resulting in the identification of T. cruzi membrane-associated proteins that are potential vaccine candidates.

scholarly journals The Protective Mechanism of Deuterated Linoleic Acid Involves the Activation of the Ca2+ Signaling System of Astrocytes in Ischemia In Vitro

2021 ◽  
Vol 22 (24) ◽  
pp. 13216
Author(s):  
Egor A. Turovsky ◽  
Elena G. Varlamova ◽  
Sergey V. Gudkov ◽  
Egor Y. Plotnikov
Keyword(s):  
Cell Death ◽  
Fatty Acid ◽  
Cortical Neurons ◽  
Apoptotic Cell ◽  
Glucose Deprivation ◽  
Protective Mechanism ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Protective Genes

Ischemia-like (oxygen-glucose deprivation, OGD) conditions followed by reoxygenation (OGD/R) cause massive death of cerebral cortex cells in culture as a result of the induction of necrosis and apoptosis. Cell death occurs as a result of an OGD-induced increase in Ca2+ ions in the cytosol of neurons and astrocytes, an increase in the expression of genes encoding proapoptotic and inflammatory genes with suppression of protective genes. The deuterated form of linoleic polyunsaturated fatty acid (D4-Lnn) completely inhibits necrosis and greatly reduces apoptotic cell death with an increase in the concentration of fatty acid in the medium. It was shown for the first time that D4-Lnn, through the activation of the phosphoinositide calcium system of astrocytes, causes their reactivation, which correlates with the general cytoprotective effect on the cortical neurons and astrocytes in vitro. The mechanism of the cytoprotective action of D4-Lnn involves the inhibition of the OGD-induced calcium ions, increase in the cytosolic and reactive oxygen species (ROS) overproduction, the enhancement of the expression of protective genes, and the suppression of damaging proteins.

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