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1.
Genes Cells ; 26(9): 727-738, 2021 Sep.
Article in English | MEDLINE | ID: mdl-34166546

ABSTRACT

CDP-diacylglycerol synthases (Cds) are conserved from bacteria to eukaryotes. Bacterial CdsA is involved not only in phospholipid biosynthesis but also in biosynthesis of glycolipid MPIase, an essential glycolipid that catalyzes membrane protein integration. We found that both Cds4 and Cds5 of Arabidopsis chloroplasts complement cdsA knockout by supporting both phospholipid and MPIase biosyntheses. Comparison of the sequences of CdsA and Cds4/5 suggests a difference in membrane topology at the C-termini, since the region assigned as the last transmembrane region of CdsA, which follows the conserved cytoplasmic domain, is missing in Cds4/5. Deletion of the C-terminal region abolished the function, indicating the importance of the region. Both 6 × His tag attachment to CdsA and substitution of the C-terminal 6 residues with 6 × His did not affect the function. These 6 × His tags were sensitive to protease added from the cytosolic side in vitro, indicating that this region is not a transmembrane one but forms a membrane-embedded reentrant loop. Thus, the C-terminal region of Cds homologues forms a reentrant loop, of which structure is important for the Cds function.


Subject(s)
Arabidopsis Proteins/chemistry , Chloroplasts/genetics , Diacylglycerol Cholinephosphotransferase/chemistry , Arabidopsis , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , Chloroplasts/metabolism , Diacylglycerol Cholinephosphotransferase/genetics , Diacylglycerol Cholinephosphotransferase/metabolism , Escherichia coli , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Genetic Complementation Test , Protein Domains
2.
Structure ; 27(8): 1258-1269.e4, 2019 08 06.
Article in English | MEDLINE | ID: mdl-31178220

ABSTRACT

In mitochondria, CDP-diacylglycerol (CDP-DAG) is a crucial precursor for cardiolipin biosynthesis. Mitochondrial CDP-DAG is synthesized by the translocator assembly and maintenance protein 41 (Tam41) through an elusive process. Here we show that Tam41 adopts sequential catalytic mechanism, and report crystal structures of the bulk N-terminal region of Tam41 from Schizosaccharomyces pombe in the apo and CTP-bound state. The structure reveals that Tam41 contains a nucleotidyltransferase (NTase) domain and a winged helix domain. CTP binds to an "L"-shaped pocket sandwiched between the two domains. Rearrangement of a loop region near the active site is essential for opening the CTP-binding pocket. Docking of phosphatidic acid/CDP-DAG in the structure suggests a lipid entry/exit pathway connected to the "L"-shaped pocket. The C-terminal region of SpTam41 contains a positively charged amphipathic helix crucial for membrane association and participates in binding phospholipids. These results provide detailed insights into the mechanism of CDP-DAG biosynthesis in mitochondria.


Subject(s)
Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/metabolism , Schizosaccharomyces/metabolism , Catalytic Domain , Cell Membrane/metabolism , Cytidine Triphosphate/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Lipid Metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Models, Molecular , Protein Conformation , Schizosaccharomyces/chemistry , Substrate Specificity
3.
Article in English | MEDLINE | ID: mdl-28944216

ABSTRACT

The facultative intracellular bacterium Legionella pneumophila proliferates within amoebae and human alveolar macrophages, and it is the causative agent of Legionnaires' disease, a life-threatening pneumonia. Within host cells, L. pneumophila establishes a replicative haven by delivering numerous effector proteins into the host cytosol, many of which target membrane trafficking by manipulating the function of Rab GTPases. The Legionella effector AnkX is a phosphocholine transferase that covalently modifies host Rab1 and Rab35. However, a detailed understanding of the biological consequence of Rab GTPase phosphocholination remains elusive. Here, we broaden the understanding of AnkX function by presenting three lines of evidence that it interferes with host endocytic recycling. First, using immunogold transmission electron microscopy, we determined that GFP-tagged AnkX ectopically produced in mammalian cells localizes at the plasma membrane and tubular membrane compartments, sites consistent with targeting the endocytic recycling pathway. Furthermore, the C-terminal region of AnkX was responsible for association with the plasma membrane, and we determined that this region was also able to bind the phosphoinositide lipids PI(3)P and PI(4)P in vitro. Second, we observed that mCherry-AnkX co-localized with Rab35, a regulator of recycling endocytosis and with major histocompatibility class I protein (MHC-I), a key immunoregulatory protein whose recycling from and back to the plasma membrane is Rab35-dependent. Third, we report that during infection of macrophages, AnkX is responsible for the disruption of endocytic recycling of transferrin, and AnkX's phosphocholination activity is critical for this function. These results support the hypothesis that AnkX targets endocytic recycling during host cell infection. Finally, we have demonstrated that the phosphocholination activity of AnkX is also critical for inhibiting fusion of the Legionella-containing vacuole (LCV) with lysosomes.


Subject(s)
Bacterial Proteins/metabolism , Diacylglycerol Cholinephosphotransferase/metabolism , Host-Pathogen Interactions/physiology , Legionella pneumophila/metabolism , Legionnaires' Disease/metabolism , Amoeba/microbiology , Animals , Ankyrin Repeat , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , COS Cells , Cell Membrane/metabolism , Chlorocebus aethiops , Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/genetics , Endocytosis/physiology , Endosomes/metabolism , HEK293 Cells , HeLa Cells , Host-Pathogen Interactions/immunology , Humans , Legionella pneumophila/enzymology , Legionella pneumophila/genetics , Legionella pneumophila/pathogenicity , Legionnaires' Disease/immunology , Lysosomes , Macrophages/microbiology , Major Histocompatibility Complex , Phosphatidylinositol Phosphates/metabolism , Phosphatidylinositols/metabolism , Phosphorylcholine/metabolism , Protein Transport , Recombinant Proteins , Vacuoles/metabolism , rab GTP-Binding Proteins/metabolism
4.
Genet Mol Res ; 15(3)2016 Aug 26.
Article in English | MEDLINE | ID: mdl-27706605

ABSTRACT

The enzymes 1,2-diacylglycerol cholinephosphotrans-ferase (CPT) and lysophosphatidylcholine acyltransferase (LPCAT) are important in lipid metabolism in soybean seeds. Thus, understand-ing the genes that encode these enzymes may enable their modification and aid the improvement of soybean oil quality. In soybean, the genes encoding these enzymes have not been completely described; there-fore, this study aimed to identify, characterize, and analyze the in silico expression of these genes in soybean. We identified two gene models encoding CPT and two gene models encoding LPCAT, one of which presented an alternative transcript. The sequences were positioned on the physical map of soybean and the promoter regions were analyzed. Cis-elements responsible for seed-specific expression and responses to biotic and abiotic stresses were identified. Virtual expression analysis of the gene models for CPT and LPCAT indicated that these genes are expressed under different stress conditions, in somatic embryos during differentiation, in immature seeds, root tissues, and calli. Putative ami-no acid sequences revealed the presence of transmembrane domains, and analysis of the cellular localization of these enzymes revealed they are located in the endoplasmic reticulum.


Subject(s)
1-Acylglycerophosphocholine O-Acyltransferase/genetics , Diacylglycerol Cholinephosphotransferase/genetics , Endoplasmic Reticulum/enzymology , Glycine max/genetics , Plant Proteins/genetics , 1-Acylglycerophosphocholine O-Acyltransferase/chemistry , 1-Acylglycerophosphocholine O-Acyltransferase/metabolism , Alternative Splicing , Amino Acid Sequence , Computer Simulation , Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/metabolism , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/ultrastructure , Gene Expression , Lipid Metabolism/genetics , Models, Genetic , Physical Chromosome Mapping , Plant Cells/enzymology , Plant Cells/ultrastructure , Plant Proteins/chemistry , Plant Proteins/metabolism , Plant Roots/cytology , Plant Roots/enzymology , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Seeds/cytology , Seeds/enzymology , Sequence Alignment , Glycine max/cytology , Glycine max/enzymology
5.
Biochemistry ; 55(31): 4375-85, 2016 08 09.
Article in English | MEDLINE | ID: mdl-27404583

ABSTRACT

The intracellular pathogen Legionella pneumophila infects lung macrophages and injects numerous effector proteins into the host cell to establish a vacuole for proliferation. The necessary interference with vesicular trafficking of the host is achieved by modulation of the function of Rab GTPases. The effector protein AnkX chemically modifies Rab1b and Rab35 by covalent phosphocholination of serine or threonine residues using CDP-choline as a donor. So far, the phosphoryl transfer mechanism and the relevance of observed autophosphocholination of AnkX remained disputable. We designed tailored caged compounds to make this type of enzymatic reaction accessible for time-resolved Fourier transform infrared difference spectroscopy. By combining spectroscopic and biochemical methods, we determined that full length AnkX is autophosphocholinated at Ser521, Thr620, and Thr943. However, autophosphocholination loses specificity for these sites in shortened constructs and does not appear to be relevant for the catalysis of the phosphoryl transfer. In contrast, transient phosphocholination of His229 in the conserved catalytic motif might exist as a short-lived reaction intermediate. Upon substrate binding, His229 is deprotonated and locked in this state, being rendered capable of a nucleophilic attack on the pyrophosphate moiety of the substrate. The proton that originated from His229 is transferred to a nearby carboxylic acid residue. Thus, our combined findings support a ping-pong mechanism involving phosphocholination of His229 and subsequent transfer of phosphocholine to the Rab GTPase. Our approach can be extended to the investigation of further nucleotidyl transfer reactions, which are currently of reemerging interest in regulatory pathways of host-pathogen interactions.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/metabolism , Legionella pneumophila/enzymology , Ankyrin Repeat , Bacterial Proteins/genetics , Biocatalysis , Catalytic Domain , Diacylglycerol Cholinephosphotransferase/genetics , Host-Pathogen Interactions , Humans , Legionella pneumophila/genetics , Legionella pneumophila/pathogenicity , Models, Molecular , Phosphorylcholine/metabolism , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectroscopy, Fourier Transform Infrared , rab GTP-Binding Proteins/metabolism , rab1 GTP-Binding Proteins/metabolism
6.
Prostaglandins Other Lipid Mediat ; 121(Pt B): 176-83, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26358846

ABSTRACT

Interleukin 1 beta (IL-1ß) induced platelet activating factor (PAF) synthesis in U-937 cells through stimulation of acetyl-CoA:lysoPAF-acetyltransferase (lyso PAF-AT) at 3 h and DTT-independentCDP-choline-1-alkyl-2-acetyl-sn-glycerol cholinophosphotransferase (PAF-CPT) at 0.5 h. The aim of this study was to investigate the effect of tyrosol (T), resveratrol (R) and their acetylated derivatives(AcDs) which exhibit enhanced bioavailability, on PAF synthesis in U-937 after IL-1ß stimulation. The specific activity of PAF enzymes and intracellular levels were measured in cell homogenates. T and R concentration capable of inducing 50% inhibition in IL-1ß effect on lyso PAF-AT was 48 µΜ ± 11 and 157 µΜ ± 77, for PAF-CPT 246 µΜ ± 61 and 294 µΜ ± 102, respectively. The same order of concentration was also observed on inhibiting PAF levels produced by IL-1ß. T was more potent inhibitor than R (p<0.05). AcDs of T retain parent compound inhibitory activity, while in the case of R only two AcDs retain the activity. The observed inhibitory effect by T,R and their AcDs, may partly explain their already reported beneficial role.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antioxidants/pharmacology , Down-Regulation/drug effects , Monocytes/drug effects , Phenylethyl Alcohol/analogs & derivatives , Platelet Activating Factor/antagonists & inhibitors , Stilbenes/pharmacology , 1-Alkyl-2-acetylglycerophosphocholine Esterase/antagonists & inhibitors , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Acetylation , Acetyltransferases/antagonists & inhibitors , Acetyltransferases/chemistry , Acetyltransferases/metabolism , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Antioxidants/chemical synthesis , Antioxidants/chemistry , Cell Line , Diacylglycerol Cholinephosphotransferase/antagonists & inhibitors , Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Monocytes/immunology , Monocytes/metabolism , Osmolar Concentration , Phenylethyl Alcohol/pharmacology , Platelet Activating Factor/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Resveratrol , Stilbenes/chemistry
7.
Dev Cell ; 21(5): 813-24, 2011 Nov 15.
Article in English | MEDLINE | ID: mdl-22075145

ABSTRACT

Polyphosphoinositides are lipid signaling molecules generated from phosphatidylinositol (PtdIns) with critical roles in vesicular trafficking and signaling. It is poorly understood where PtdIns is located within cells and how it moves around between membranes. Here we identify a hitherto-unrecognized highly mobile membrane compartment as the site of PtdIns synthesis and a likely source of PtdIns of all membranes. We show that the PtdIns-synthesizing enzyme PIS associates with a rapidly moving compartment of ER origin that makes ample contacts with other membranes. In contrast, CDP-diacylglycerol synthases that provide PIS with its substrate reside in the tubular ER. Expression of a PtdIns-specific bacterial PLC generates diacylglycerol also in rapidly moving cytoplasmic objects. We propose a model in which PtdIns is synthesized in a highly mobile lipid distribution platform and is delivered to other membranes during multiple contacts by yet-to-be-defined lipid transfer mechanisms.


Subject(s)
Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Organelles/metabolism , Phosphatidylinositols/biosynthesis , Phosphatidylinositols/metabolism , Animals , COS Cells , Cell Membrane/chemistry , Chlorocebus aethiops , Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/metabolism , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/enzymology , HEK293 Cells , Humans , Organelles/chemistry , Phosphatidylinositols/chemistry , Signal Transduction
8.
Int J Parasitol ; 40(11): 1257-68, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20385136

ABSTRACT

Cytidine diphosphate diacylglycerol synthase (CDS) diverts phosphatidic acid towards the biosynthesis of CDP-DAG, an obligatory liponucleotide intermediate in anionic phospholipid biosynthesis. The 78kDa predicted Plasmodium falciparum CDS (PfCDS) is recovered as a 50 kDa conserved C-terminal cytidylyltransferase domain (C-PfCDS) and a 28kDa fragment that corresponds to the unusually long hydrophilic asparagine-rich N-terminal extension (N-PfCDS). Here, we show that the two fragments of PfCDS are the processed forms of the 78 kDa pro-form that is encoded from a single transcript with no alternate translation start site for C-PfCDS. PfCDS, which shares 54% sequence identity with Plasmodium knowlesi CDS (PkCDS), could substitute for PkCDS in P. knowlesi. Experiments to disrupt either the full-length or the N-terminal extension of PkCDS indicate that not only the C-terminal cytidylyltransferase domain but also the N-terminal extension is essential to Plasmodium spp. PkCDS and PfCDS introduced in P. knowlesi were processed in the parasite, suggesting a conserved parasite-dependent mechanism. The N-PfCDS appears to be a peripheral membrane protein and is trafficked outside the parasite to the parasitophorous vacuole. Although the function of this unusual N-PfCDS remains enigmatic, the study here highlights features of this essential gene and its biological importance during the intra-erythrocytic cycle of the parasite.


Subject(s)
Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/metabolism , Plasmodium falciparum/enzymology , Plasmodium knowlesi/enzymology , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Amino Acid Sequence , Animals , Chlorocebus aethiops , Cytidine Diphosphate Diglycerides/biosynthesis , Diacylglycerol Cholinephosphotransferase/genetics , Erythrocytes/parasitology , Humans , Malaria/parasitology , Plasmodium falciparum/chemistry , Plasmodium falciparum/genetics , Plasmodium falciparum/growth & development , Plasmodium knowlesi/chemistry , Plasmodium knowlesi/genetics , Plasmodium knowlesi/growth & development , Protein Structure, Tertiary , Protozoan Proteins/genetics
9.
Curr Microbiol ; 58(6): 535-40, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19219498

ABSTRACT

A gene, mf1, encoding a novel cholinephosphotransferase in glycoglycerophospholipid (GGPL) biosynthesis of Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, and expressed in Escherichia coli. The mf1 gene comprises an open reading frame of 777 bp encoding 258 amino acids. The mf1 gene product, Mf1, has 23% amino acid homology with LicD of Haemophilus influenzae but no homology with genes of other Mycoplasma species in the GenBank database. The reaction product of Mf1 using alpha-glucopyranosyl-1,2-dipalmitoilglycerol and cytidine 5'-diphosphocholine (CDP-choline) as substrates showed the specific protonated molecule at m/z 896, which corresponded to GGPL-I as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem mass spectrometry (MS) analysis of protonated molecules at m/z 896. These results identified mf1 as a novel cholinephosphotransferase and showed that the phosphocholine transfer step is involved in the GGPL biosynthesis pathway of M. fermentans. This is the first report of a GGPL biosynthesis enzyme.


Subject(s)
Bacterial Proteins/metabolism , Cloning, Molecular , Diacylglycerol Cholinephosphotransferase/metabolism , Gene Expression , Glycerophosphates/biosynthesis , Mycoplasma fermentans/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/genetics , Glycerophosphates/chemistry , Molecular Sequence Data , Mycoplasma fermentans/chemistry , Mycoplasma fermentans/classification , Mycoplasma fermentans/genetics , Phylogeny , Substrate Specificity
10.
Biochem Biophys Res Commun ; 312(4): 1104-10, 2003 Dec 26.
Article in English | MEDLINE | ID: mdl-14651986

ABSTRACT

Cholinephosphotransferase (CPT), the terminal enzyme in the de novo synthesis of phosphatidylcholine (PC), has an important role in regulating the acyl group of PC in mammalian cells. A 593bp cDNA coding for the 3(')-end of the CPT gene has been cloned from guinea pig liver using degenerative oligos based on the human CPT gene. It has 85% amino acid homology with the human CPT enzyme and amino acid variations were found to cluster at few points. Restriction enzyme polymorphisms were found particularly with respect to BamHI and NcoI. Hydrophobic and helix plot analysis of the sequence shows a similar pattern to human counterpart except for amino acid residues 142-179 and 173-179. PCR analysis suggested that a predominant pseudogene may be present in guinea pig and also the intronic sequences were much shorter when compared to the human CPT gene. We are the first to report on the C-terminal 195 amino acid residues of the CPT gene from any animal species alike in many aspects of cellular metabolism. The probable differences in genomic organization and its expression in different cancer cells have been discussed here having CPT as an important target for cancer drug development.


Subject(s)
Cloning, Molecular/methods , Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/genetics , Sequence Alignment , Amino Acid Sequence , Animals , Base Sequence , Humans , Molecular Sequence Data , Molecular Weight , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sequence Analysis, Protein , Sequence Homology , Species Specificity
11.
Mol Biochem Parasitol ; 121(1): 87-98, 2002 Apr 30.
Article in English | MEDLINE | ID: mdl-11985865

ABSTRACT

CDP-diacylglycerol synthase (CDS) is a key rate-limiting enzyme in the phospholipid metabolism of Plasmodium falciparum, converting phosphatidic acid to CDP-diacylglycerol. The CDS gene is predominantly expressed in the mature intraerythrocytic stages. Consequently, we physically and functionally characterized the CDS gene promoter. The mRNA transcription initiation site was mapped 121 bp upstream of the CDS gene translation start site. A 1909 bp 5' upstream sequence was isolated and found to be transcriptionally active thus constituting a functional CDS promoter. Mapping of this promoter identified a 44 bp cis-acting sequence, located between -1640 and -1596 bp upstream of the ATG codon, essential for efficient transcriptional activity. This 44 bp sequence binds specifically to nuclear factors from trophozoite stage parasites. We further showed that a 24 bp element, lying within the 44 bp sequence, mediates the specific binding to nuclear proteins and shows no significant homology to known eukaryotic DNA consensus sequence elements that bind transcription factors. The deletion of the 24 bp element abrogated promoter activity, indicating that this cis-acting sequence element is essential for efficient transcription of the CDS gene.


Subject(s)
Diacylglycerol Cholinephosphotransferase/genetics , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Enzymologic , Plasmodium falciparum/enzymology , Promoter Regions, Genetic/genetics , Transcription, Genetic , Animals , Base Sequence , Codon, Initiator , Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/metabolism , Gene Deletion , Humans , Molecular Sequence Data , Nuclear Proteins/metabolism , Plasmodium falciparum/genetics
12.
J Biol Chem ; 273(22): 13482-7, 1998 May 29.
Article in English | MEDLINE | ID: mdl-9593682

ABSTRACT

Cholinephosphotransferase (EC 2.7.8.2) catalyzes the formation of a phosphoester bond via the transfer of a phosphocholine moiety from CDP-choline to diacylglycerol forming phosphatidylcholine and releasing CMP. A motif, Asp113-Gly114-(X)2-Ala117-Arg118-(X)8-Gly127+ ++-(X)3-Asp131-(X)3-Asp135, located within the CDP-choline binding region of Saccharomyces cerevisiae cholinephosphotransferase (CPT1 ?/Author: Please confirm that a gene is meant here.) is also found in several other phospholipid synthesizing enzymes that catalyze the formation of a phosphoester bond utilizing a CDP-alcohol and a second alcohol as substrates. To determine if this motif is diagnostic of the above reaction type scanning alanine mutagenesis of the conserved residues within S. cerevisiae cholinephosphotransferase was performed. Enzyme activity was assessed in vitro using a mixed micelle enzyme assay and in vivo by determining the ability of the mutant enzymes to restore phosphatidylcholine synthesis from radiolabeled choline in an S. cerevisiae strain devoid of endogenous cholinephosphotransferase activity. Alanine mutants of Gly114, Gly127, Asp131, and Asp135 were inactive; mutants, Ala117 and Arg118 displayed reduced enzyme activity, and Asp113 displayed wild type activity. The analysis described is the first molecular characterization of a CDP-alcohol phosphotransferase motif and results predict a catalytic role utilizing a general base reaction proceeding through Asp131 or Asp135 via a direct nucleophilic attack of the hydroxyl of diacylglyerol on the phosphoester bond of CDP-choline that does not proceed via an enzyme bound intermediate. Residues Ala117 and Arg118 do not participate directly in catalysis but are likely involved in substrate binding or positioning with Arg118 predicted to associate with a phosphate moiety of CDP-choline.


Subject(s)
Diacylglycerol Cholinephosphotransferase/genetics , Saccharomyces cerevisiae/enzymology , Alanine/chemistry , Alanine/genetics , Amino Acid Sequence , Amino Acid Substitution , Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/metabolism , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed
13.
Biochim Biophys Acta ; 1348(1-2): 100-10, 1997 Sep 04.
Article in English | MEDLINE | ID: mdl-9370321

ABSTRACT

Cholinephosphotransferase transfers a phosphocholine moiety from CDP-choline to diacylglycerol thus forming phosphatidylcholine (PtdCho) and CMP. This reaction defines the ultimate step in the Kennedy pathway for the genesis of de novo synthesized PtdCho. Hence, the intracellular location of cholinephosphotransferase identifies both the site from which de novo synthesized PtdCho is transported to other organelles and the site from which it is assembled with proteins and other lipids for secretion from the cell during the generation of lung surfactant, lipoproteins, and bile. Most subcellular fractionation studies observed the majority of cholinephosphotransferase activity in the endoplasmic reticulum, although the method of subcellular fractionation was found to grossly affect these results with activity alternately dispersed within Golgi, nuclear, and mitochondrial fractions. Coupling subcellular fractionation results with immunofluorescence or electron microscopy studies would resolve the issue of the site of PtdCho synthesis. However, antibodies have yet to be generated to cholinephosphotransferase since its integral membrane-bound nature has prevented its purification from any source and a mammalian cholinephosphotransferase cDNA has also yet to be isolated. However, cholinephosphotransferase genes have recently been isolated from the yeast Saccharomyces cerevisiae. Structure/function analysis of the S. cerevisiae cholinephosphotransferase has allowed for an in depth molecular examination resulting in the identification of the catalytic site. In addition, this analysis has generated the predicted amino acid data necessary to produce antibodies to pursue the site of PtdCho synthesis in this organism, as well as to provide information that should allow for the isolation of mammalian cholinephosphotransferase cDNA(s).


Subject(s)
Diacylglycerol Cholinephosphotransferase/metabolism , Amino Acid Sequence , Cloning, Molecular , Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/genetics , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino Acid , Substrate Specificity
14.
Biochim Biophys Acta ; 1348(1-2): 166-72, 1997 Sep 04.
Article in English | MEDLINE | ID: mdl-9370329

ABSTRACT

CDP-diacylglycerol resides at the branch point of glycerolipid biosynthesis as precursor of both the phosphoinositides and phosphatidylglycerol. The discovery of the phosphoinositide signal transduction pathway and the recognition of its prominent role in intracellular communication has focused new attention on CDP-diacylglycerol synthase. As a rate-limiting step in this pathway, it is a likely target for regulation. Exploration of this possibility will be facilitated by the recent cloning of mammalian CDP-DAG synthase.


Subject(s)
Diacylglycerol Cholinephosphotransferase/metabolism , Animals , Cloning, Molecular , Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/genetics , Protein Conformation , Substrate Specificity
15.
J Biol Chem ; 272(14): 9503-9, 1997 Apr 04.
Article in English | MEDLINE | ID: mdl-9083091

ABSTRACT

A cDNA encoded a 462-amino acid protein, which showed CDP-diacylglycerol synthase (CDS) activity was cloned for the first time as the vertebrate enzyme molecule from rat brain cDNA library. The deduced molecular mass of this rat CDS was 53 kDa, and putative primary structure included several possible membrane- spanning regions. At the amino acid sequence level, rat CDS shared 55.5%, 31. 7%, and 20.9% identity with already known Drosophila, Saccharomyces cerevisiae, and Escherichia coli CDS, respectively. This rat CDS preferred 1-stearoyl-2-arachidonoyl phosphatidic acid as a substrate, and its activity was strongly inhibited by phosphatidylglycerol 4, 5-bisphosphate. By immunoblotting analysis of COS cells overexpressed with the epitope-tagged for rat CDS, a 60-kDa band was detected. By epitope-tag immunocytochemistry, the CDS protein was mainly localized in close association with the membrane of the endoplasmic reticulum of the transfected cells. The intense mRNA expression of CDS was localized in the cerebellar Purkinje cells, the pineal body, and the inner segment of photoreceptor cells. Additionally, very intense expression was detected in postmitotic spermatocytes and spermatids.


Subject(s)
Brain/enzymology , Diacylglycerol Cholinephosphotransferase/genetics , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Diacylglycerol Cholinephosphotransferase/chemistry , In Situ Hybridization , Male , Molecular Sequence Data , Phosphatidylinositol 4,5-Diphosphate/metabolism , RNA, Messenger/metabolism , Rats , Rats, Wistar , Retina/enzymology , Testis/enzymology
16.
J Neurochem ; 67(5): 2200-3, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8863531

ABSTRACT

A critical step in the supply of substrate for the phosphoinositide signal transduction pathway is the formation of the liponucleotide intermediate, CDP-diacylglycerol, catalyzed by CDP-diacylglycerol synthase. Further insight into the regulation of phosphoinositide biosynthesis was sought by cloning of the gene for the vertebrate enzyme. Sequence of the corresponding gene from Drosophila was used to prepare a probe for screening of a human neuronal cell cDNA library. A cDNA was isolated with a predicted open reading frame of 1,332 bases, encoding a protein of 51 kDa. The amino acid sequence showed 50% identity (75% similarity) to that of Drosophila eye CDP-diacylglycerol synthase and substantial similarity to the Saccharomyces cerevisiae and Escherichia coli homologues. Northern blot analysis, with human cDNA riboprobes, suggested that the corresponding mRNA was expressed in all human tissues examined. Expression of the human cDNA in COS cells resulted in a more than fourfold increase in CDP-diacylglycerol synthase activity. Knowledge of the sequence of vertebrate CDP-diacylglycerol synthase should facilitate further investigations into its regulation and the possible existence of distinct isoforms.


Subject(s)
Diacylglycerol Cholinephosphotransferase/biosynthesis , Diacylglycerol Cholinephosphotransferase/chemistry , Neurons/enzymology , Amino Acid Sequence , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Cloning, Molecular , Drosophila/enzymology , Escherichia coli/enzymology , Humans , Molecular Sequence Data , Organ Specificity , RNA, Messenger/biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Sequence Homology, Amino Acid , Transcription, Genetic , Transfection
17.
Comp Biochem Physiol B Biochem Mol Biol ; 112(3): 493-501, 1995 Nov.
Article in English | MEDLINE | ID: mdl-8529026

ABSTRACT

While steady-state kinetic parameters (metabolite pools, Km and activation energies) are partially known for the enzymes involved in phosphatidylcholine synthesis and degradation in mammalian brain, they are not available for the nervous system of lower vertebrates or invertebrates. Since the extent of evolutionary development of an enzyme is not known a priori, we evaluated the kinetic and thermodynamic parameters of choline kinase, CTP:phosphocholine cytidylyltransferase, choline phosphotransferase and glycerophosphorylcholine phosphodiesterase in squid (Loligo pealei) optic lobe, dogfish (Mustelus canis) and rat brain. For all these enzyme activities, basic similarities in Km and inhibitor effect were found. The same was true for the activation energies Ea, with the exception of squid choline kinase and dogfish cytidylyltransferase. Treatment of microsomal membranes with phospholipase C sharply inhibited cytidylyltransferase activity in all three animal species. In dogfish brain, glycerophosphorylcholine phosphodiesterase activity was undetectable. Our results are consistent with the notion that the kinetic properties of the enzyme activities leading to the preservation of the phosphatidylcholine membranous pool may have appeared early in metazoan evolution and been fully conserved in mammals.


Subject(s)
Biological Evolution , Brain/enzymology , Decapodiformes/metabolism , Dogfish/metabolism , Optic Lobe, Nonmammalian/enzymology , Phosphatidylcholines/metabolism , Animals , Choline Kinase/antagonists & inhibitors , Choline Kinase/chemistry , Choline Kinase/metabolism , Choline-Phosphate Cytidylyltransferase , Diacylglycerol Cholinephosphotransferase/antagonists & inhibitors , Diacylglycerol Cholinephosphotransferase/chemistry , Enzyme Activation , Enzyme Inhibitors/pharmacology , Kinetics , Nucleotidyltransferases/antagonists & inhibitors , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/metabolism , Rats , Thermodynamics , Type C Phospholipases/pharmacology
18.
Biochim Biophys Acta ; 1170(2): 157-64, 1993 Oct 13.
Article in English | MEDLINE | ID: mdl-8399340

ABSTRACT

The synthesis of platelet-activating factor (PAF) was studied in microsomal fractions of cerebral cortices of 15-day-old rabbits. These included: a total microsomal fraction P3, rough and smooth microsomes, R and S, and microsomal fraction P derived from isolated nerve cell bodies. Cholinephosphotransferase (CPT) generating PAF from alkylacetylglycerol had the highest specific activities in fractions R and P (24 and 6 times the homogenate values, based on membrane phospholipid content). This CPT activity differed from that which synthesized phosphatidylcholine as the latter was sensitive to dithiothreitol inhibition and was more readily inhibited by Triton X-100. As the CPT activity for PAF synthesis relies on the production of alkylacetylglycerol we studied the acetyltransferase which forms 1-alkyl-2-acetyl-sn-glycero-3-phosphate (AAGP). This enzyme had the highest specific activity in fraction R, followed by fractions P3 and P. There was evidence that the acetyltransferase was more active in a phosphorylated form. NaF maximized the recovery of AAGP products in the assays. The pH optimum for acetylation was in a range of 8.0-9.0. Lyso PAF did not inhibit the formation of AAGP and the rates of formation of PAF by acetylation were less than 5% of values for AAGP synthesis. During AAGP formation there was no evidence for subsequent alkylacetylglycerol formation in the absence of NaF, but a small formation of radioactive PAF could be demonstrated from AAGP under the CPT assay conditions.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Acetyltransferases/chemistry , Brain Chemistry , Diacylglycerol Cholinephosphotransferase/chemistry , Platelet Activating Factor/biosynthesis , Animals , Cerebral Cortex/enzymology , Cytidine Diphosphate Choline/pharmacology , Dithiothreitol/pharmacology , Glyceryl Ethers/pharmacology , Microsomes/enzymology , Phosphatidylcholines/biosynthesis , Platelet Activating Factor/analogs & derivatives , Platelet Activating Factor/pharmacology , Rabbits , Sodium Fluoride/pharmacology
19.
Lipids ; 28(2): 89-96, 1993 Feb.
Article in English | MEDLINE | ID: mdl-8382767

ABSTRACT

The solubilization and partial purification of cholinephosphotransferase (CDPcholine:1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2) from rat liver microsomes were examined in the presence of ionic (sodium deoxycholate), nonionic (Triton X-100, n-octylglycoside), or zwitter ionic (CHAPS) detergents. Among the four detergents tested, only sodium deoxycholate was found to be an efficient solubilizer of cholinephosphotransferase activity from microsomal membranes, whereas the other three detergents caused irreversible inactivation of the enzyme at the solubilization step. Addition of phospholipids at the solubilization step, or after solubilization of the membrane proteins, could not preserve or reconstitute activity to any extent. The sodium deoxycholate-solubilized activity was partially purified by gel permeation chromatography (Superose 12HR). The partially purified preparation appeared to consist of a large aggregate containing phospholipids; further dissociation of the protein-phospholipid complex caused complete inactivation of the enzyme. The partially purified cholinephosphotransferase showed a specific activity of 100-130 nmol/min/mg protein, which is the highest activity reported to date from any tissue source; this amounts to a 4-fold enrichment of cholinephosphotransferase activity from the original KCl-washed rat liver microsomes. Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) activity was copurified and 6-fold enriched with a total recovery of 60%. During the purification of cholinephosphotransferase activity, a putative endogenous inhibitor of cholinephosphotransferase was also solubilized and was isolated from the microsomal membranes. This heat-labile, nondialyzable inhibitor was shown to act specifically on cholinephosphotransferase and not on ethanolaminephosphotransferase. Further characterization of the inhibitory activity revealed that it may act at the binding step of the cholinephosphotransferase to its lipid substrate, diacylglycerol.


Subject(s)
Diacylglycerol Cholinephosphotransferase/chemistry , Microsomes, Liver/enzymology , Animals , Chromatography, Gel , Deoxycholic Acid , Detergents/pharmacology , Diacylglycerol Cholinephosphotransferase/antagonists & inhibitors , Diacylglycerol Cholinephosphotransferase/isolation & purification , Dialysis , Diglycerides/pharmacology , Electrophoresis, Polyacrylamide Gel , Female , Hydrogen-Ion Concentration , Magnesium/pharmacology , Male , Manganese/pharmacology , Octoxynol , Polyethylene Glycols/pharmacology , Potassium Chloride/pharmacology , Rats , Rats, Wistar , Solubility
20.
Mol Cell Biochem ; 101(2): 157-66, 1991 Mar 13.
Article in English | MEDLINE | ID: mdl-1650426

ABSTRACT

We have reported earlier that cholinephosphotransferase (EC 2.7.8.2) is present in both mitochondria and microsomes of fetal guinea pig lung. This study was designed to compare the properties of mitochondrial and microsomal cholinephosphotransferase in fetal guinea pig lung. Various parameters, such as substrate specificity, Km values, sensitivity to N-ethylmaleimide, dithiothreitol and trypsin were measured. Both showed significant preference for unsaturated diacylglycerols over saturated diacylglycerols. Data on Km and Vmax indicate that the affinity of this enzyme for different diacylglycerols varies between the two forms. The ID50 values for N-ethylmaleimide were 20 mM and 12.5 mM for the mitochondrial and microsomal form of the enzyme, respectively. Dithiothreitol showed an inhibitory effect on both; however, the mitochondrial form was inhibited less than the microsomal form. The effects of N-ethylmaleimide and dithiothreitol on both forms of enzyme indicated that the microsomal cholinephosphotransferase requires a higher concentration of -SH for its activity than the mitochondrial enzyme does. The enzyme was inhibited by trypsin in both mitochondria and microsome under isotonic condition suggesting that this enzyme is on the outside of the membrane in both endoplasmic reticulum and mitochondria.


Subject(s)
Diacylglycerol Cholinephosphotransferase/chemistry , Lung/enzymology , Microsomes/enzymology , Mitochondria/enzymology , Animals , Diacylglycerol Cholinephosphotransferase/metabolism , Diglycerides/metabolism , Female , Fetus , Guinea Pigs , Pregnancy , Substrate Specificity
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