scholarly journals Bifunctional CTP:Inositol-1-Phosphate Cytidylyltransferase/CDP-Inositol:Inositol-1-Phosphate Transferase, the Key Enzyme for Di-myo-Inositol-Phosphate Synthesis in Several (Hyper)thermophiles

2007 ◽  
Vol 189 (15) ◽  
pp. 5405-5412 ◽  
Author(s):  
Marta V. Rodrigues ◽  
Nuno Borges ◽  
Mafalda Henriques ◽  
Pedro Lamosa ◽  
Rita Ventura ◽  
...  
Keyword(s):  
Pyrococcus Furiosus ◽  
Inositol Phosphate ◽  
Thermotoga Maritima ◽  
Single Gene ◽  
Cloning And Expression ◽  
Aquifex Aeolicus ◽  
Inositol 1 ◽  
Cell Extracts ◽  
Genomic Approach ◽  
Key Enzyme

ABSTRACT The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of Archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol. 188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in Escherichia coli of the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3′-phosphate-1′-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus, Pyrococcus furiosus, Thermococcus kodakaraensis, Aquifex aeolicus, and Rubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP and l-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol and l-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP and l-myo-inositol-1-P; the DIPP synthase domain used only l-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted in Aeropyrum pernix, Thermotoga maritima, and Hyperthermus butylicus. Additionally, using l-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3′-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.

scholarly journals A Unique β-1,2-Mannosyltransferase of Thermotoga maritima That Uses Di-myo-Inositol Phosphate as the Mannosyl Acceptor

2009 ◽  
Vol 191 (19) ◽  
pp. 6105-6115 ◽  
Author(s):  
Marta V. Rodrigues ◽  
Nuno Borges ◽  
Carla P. Almeida ◽  
Pedro Lamosa ◽  
Helena Santos
Keyword(s):  
Inositol Phosphate ◽  
Thermotoga Maritima ◽  
Isotopic Labeling ◽  
Maximal Activity ◽  
Functional Expression ◽  
Single Step ◽  
Aquifex Aeolicus ◽  
Mannose Residue ◽  
Organic Solute ◽  
Total Pool

ABSTRACT In addition to di-myo-inositol-1,3′-phosphate (DIP), a compatible solute widespread in hyperthermophiles, the organic solute pool of Thermotoga maritima comprises 2-(O-β-d-mannosyl)-di-myo-inositol-1,3′-phosphate (MDIP) and 2-(O-β-d-mannosyl-1,2-O-β-d-mannosyl)-di-myo-inositol-1,3′-phosphate (MMDIP), two newly identified β-1,2-mannosides. In cells grown under heat stress, MDIP was the major solute, accounting for 43% of the total pool; MMDIP and DIP accumulated to similar levels, each corresponding to 11.5% of the total pool. The synthesis of MDIP involved the transfer of the mannosyl group from GDP-mannose to DIP in a single-step reaction catalyzed by MDIP synthase. This enzyme used MDIP as an acceptor of a second mannose residue, yielding the di-mannosylated compound. Minor amounts of the tri-mannosylated form were also detected. With a genomic approach, putative genes for MDIP synthase were identified in the genome of T. maritima, and the assignment was confirmed by functional expression in Escherichia coli. Genes with significant sequence identity were found only in the genomes of Thermotoga spp., Aquifex aeolicus, and Archaeoglobus profundus. MDIP synthase of T. maritima had maximal activity at 95°C and apparent Km values of 16 mM and 0.7 mM for DIP and GDP-mannose, respectively. The stereochemistry of MDIP was characterized by isotopic labeling and nuclear magnetic resonance (NMR): DIP selectively labeled with carbon 13 at position C1 of the l-inositol moiety was synthesized and used as a substrate for MDIP synthase. This β-1,2-mannosyltransferase is unrelated to known glycosyltransferases, and within the domain Bacteria, it is restricted to members of the two deepest lineages, i.e., the Thermotogales and the Aquificales. To our knowledge, this is the first β-1,2-mannosyltransferase characterized thus far.

Cloning and expression of a cDNA that comprises part of the gene coding for a spore coat protein of Dictyostelium discoideum

1984 ◽  
Vol 4 (11) ◽  
pp. 2273-2278
Author(s):  
B C Dowds ◽  
W F Loomis
Keyword(s):  
Dictyostelium Discoideum ◽  
Cdna Clone ◽  
Single Gene ◽  
Polyclonal Antiserum ◽  
Cloning And Expression ◽  
Spore Coat ◽  
Coat Proteins ◽  
Developmentally Regulated ◽  
Gene Coding ◽  
Spore Coat Proteins

The three major spore coat proteins of Dictyostelium discoideum are developmentally regulated, cell-type-specific proteins. They are packaged in prespore vesicles and then secreted to form the outer layer of spore coats. We have isolated a cDNA clone from the gene coding for one of these proteins, SP96, a glycoprotein of 96,000 daltons. We screened the cDNA bank by the method of hybrid select translation followed by immunoprecipitation of the translation products with SP96-specific polyclonal antiserum. We found that the gene was first transcribed into stable mRNA a few hours before the time of detection of SP96 synthesis and that the mRNA, like the protein, accumulated specifically in prespore cells and spores. SP96 constituted the same proportion of newly synthesized protein as the proportion of its message in polyadenylated RNA. SP96 appeared to be encoded by a single gene as judged by Southern blot analysis of digested genomic DNA hybridized to the cDNA clone.

Cloning and expression of a gene encoding a bacterial enzyme for aminopeptidase P from Thermotoga maritima

2000 ◽  
Vol 28 (5) ◽  
pp. A320-A320
Author(s):  
P. Selvakumar ◽  
S. Nirasawa ◽  
C. Aoyagi ◽  
S. Mori ◽  
M. Kitaoka ◽  
...  
Keyword(s):  
Thermotoga Maritima ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Bacterial Enzyme ◽  
Aminopeptidase P

Gene Cloning and Expression of a Bacteroides Gingivalis-Specific Protein Antigen in Escherichia Coli

1988 ◽  
Vol 2 (2) ◽  
pp. 310-314 ◽  
Author(s):  
Y. Abiko ◽  
M. Hayakawa ◽  
H. Aoki ◽  
H. Takiguchi
Keyword(s):  
Escherichia Coli ◽  
Specific Protein ◽  
Phage Clone ◽  
Cloning And Expression ◽  
Protein Antigen ◽  
Periodontal Pathogen ◽  
Gene Cloning And Expression ◽  
Chromosomal Dna ◽  
Cell Extracts ◽  
Bacteroides Gingivalis

Gene banks of chromosomal DNA from Bacteroides gingival is 381 were constructed utilizing the bacteriophage replacement vector λCharon4A. A clone encoding a protein antigen from B. gingivalis was identified by Western-blot screening, with use of antiserum induced to extracts of B. gingivalis cells. DNA fragments from the phage clone were subcloned into the plasmid vector pACYC184 to yield an immunoreactive clone. Cell extracts from the subclone reacted with antiserum against B. gingivalis, but did not react with antisera to B. asaccharolyticus, B. intermedius, or B. melaninogenicus. The antiserum against the purified clone products reacted with N-lauryl sarcosine extracts from B. gingivalis cells, but did not react with those of other Bacteroides cells. In addition, human serum from periodontitis patients reacted with the clone product by Western electrophoretic transfer and immunoblotting analysis. These data suggest that the gene coding for a B. gingivalis-specific protein antigen was successfully cloned and functionally expressed in Escherichia coli. This clone product may prove useful for further studies of B. gingival is as a periodontal pathogen.

The structure ofAquifex aeolicusFtsH in the ADP-bound state reveals aC2-symmetric hexamer

2015 ◽  
Vol 71 (6) ◽  
pp. 1307-1318 ◽  
Author(s):  
Marina Vostrukhina ◽  
Alexander Popov ◽  
Elena Brunstein ◽  
Martin A. Lanz ◽  
Renato Baumgartner ◽  
...  
Keyword(s):  
Active Site ◽  
Thermotoga Maritima ◽  
Adenosine Diphosphate ◽  
Crystal Form ◽  
Bound State ◽  
Aquifex Aeolicus ◽  
Protein Activity ◽  
Quadruple Mutant ◽  
Membrane Bound ◽  
Open Issues

The crystal structure of a truncated, soluble quadruple mutant of FtsH fromAquifex aeolicuscomprising the AAA and protease domains has been determined at 2.96 Å resolution in space groupI222. The protein crystallizes as a hexamer, with the protease domain forming layers in theabplane. Contacts between these layers are mediated by the AAA domains. These are highly disordered in one crystal form, but are clearly visible in a related form with a shortercaxis. Here, adenosine diphosphate (ADP) is bound to each subunit and the AAA ring exhibits twofold symmetry. The arrangement is different from the ADP-bound state of an analogously truncated, soluble FtsH construct fromThermotoga maritima. The pore is completely closed and the phenylalanine residues in the pore line a contiguous path. The protease hexamer is very similar to those described for other FtsH structures. To resolve certain open issues regarding a conserved glycine in the linker between the AAA and protease domains, as well as the active-site switch β-strand, mutations have been introduced in the full-length membrane-bound protein. Activity analysis of these point mutants reveals the crucial importance of these residues for proteolytic activity and is in accord with previous interpretation of the active-site switch and the importance of the linker glycine residue.

Oxytocin and vasopressin stimulate inositol phosphate production in human gestational myometrium and decidua cells

1986 ◽  
Vol 6 (7) ◽  
pp. 613-619 ◽  
Author(s):  
Michael P. Schrey ◽  
Alison M. Read ◽  
Philip J. Steer
Keyword(s):  
Arachidonic Acid ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Diacylglycerol Lipase ◽  
Inositol 1 ◽  
Inositol Phosphate Production ◽  
Acid Accumulation ◽  
Free Arachidonic Acid ◽  
Hydrolysis Of ◽  
Related Increase

The involvement of phosphoinositide hydrolysis in the action of oxytocin and vasopressin on the uterus was investigated in gestational myometrium and decidua cells by measuring the production of inositol phosphates. Both peptides stimulated a dose related increase in all three inositol phosphates in myometrium. This may be related to the control of sarcoplasmic Ca++ levels in the myometrium. Oxytocin and vasopressin also stimulated inositol 1-phosphate (IP) production in decidua cells. The hydrolysis of phosphatidylinositol by decidua homogenates exhibited a precursor-product relationship for diacylglycerol and arachidonic acid accumulation. Hence both peptides may mobilise free arachidonic acid, for prostaglandin biosynthesis, from decidua cell phosphoinositides by the sequential action of phospholipase C and diacylglycerol lipase.

scholarly journals Key Aromatic-Ring-Cleaving Enzyme, Protocatechuate 3,4-Dioxygenase, in the Ecologically Important MarineRoseobacter Lineage

2000 ◽  
Vol 66 (11) ◽  
pp. 4662-4672 ◽  
Author(s):  
Alison Buchan ◽  
Lauren S. Collier ◽  
Ellen L. Neidle ◽  
Mary Ann Moran
Keyword(s):  
Aromatic Compounds ◽  
Southern Hybridization ◽  
Ring Cleavage ◽  
Degenerate Pcr ◽  
Cell Extracts ◽  
Aromatic Compound Degradation ◽  
Genes Encoding ◽  
Cleavage Enzyme ◽  
Key Enzyme ◽  
Dioxygenase Activity

ABSTRACT Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the α-proteobacteria was investigated. Initial screens suggested that isolates in theRoseobacter lineage can degrade aromatic compounds via the β-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six Roseobacter isolates were screened for the presence of protocatechuate 3,4-dioxygenase, a key enzyme in the β-ketoadipate pathway. All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, benzoate, p-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate). Four of the Roseobacter group isolates had inducible protocatechuate 3,4-dioxygenase activity in cell extracts when grown onp-hydroxybenzoate. The pcaGH genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates,Sagittula stellata E-37 and isolate Y3F, and in both cases the genes could be expressed in Escherichia coli to yield dioxygenase activity. Additional genes involved in the protocatechuate branch of the β-ketoadipate pathway (pcaC,pcaQ, and pobA) were found to cluster withpcaGH in these two isolates. Pairwise sequence analysis of the pca genes revealed greater similarity between the twoRoseobacter group isolates than between genes from eitherRoseobacter strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of pcaH from two additionalRoseobacter group isolates, and Southern hybridization indicated the presence of pcaH in the remaining two isolates. This evidence of protocatechuate 3,4-dioxygenase and the β-ketoadipate pathway was found in all six Roseobacterisolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.

scholarly journals Gene and cDNA cloning and characterization of the mouse V3/V1b pituitary vasopressin receptor

1999 ◽  
Vol 22 (3) ◽  
pp. 251-260 ◽  
Author(s):  
MA Ventura ◽  
P Rene ◽  
Y de Keyzer ◽  
X Bertagna ◽  
E Clauser
Keyword(s):  
Inositol Phosphate ◽  
Rank Order ◽  
Single Gene ◽  
Dependent Manner ◽  
Binding Studies ◽  
V1b Receptor ◽  
Competition Binding ◽  
Receptor Cdna ◽  
Dose Dependent

The gene of the mouse V3/V1b receptor was identified by homology cloning. One of the genomic clones contained the entire coding sequence. The cDNA presented high identity with rat (92%) and human (84%) sequences. Southern blot analysis indicated the existence of a single gene. Tissue distribution was studied by RT-PCR. The major site of expression was the pituitary. A faint signal was also present in hypothalamus, brain, adrenal, pancreas and colon. The mouse corticotroph cell line, AtT20, did not express the transcript. In order to confirm the identity of the sequence, the V3/V1b receptor cDNA was cloned and stably expressed in CHO-AA8 Tet-Off cells under the control of tetracycline. When transfected cells were treated with arginine vasopressin (AVP), inositol phosphate production increased in a dose-dependent manner, indicating that the V3/V1b receptor couples to phospholipase C. Moreover, AVP did not stimulate cAMP production. Binding studies with [3H]AVP indicated that the affinity of the mouse V3/V1b receptor (Kd=0.5 nM) is similar to that reported for rat and human receptors. The rank order of potency established in competition binding experiments with different analogues was representative of a V3/V1b profile, distinct from V1a and V2. However, significant differences were found between human and mouse receptors tested in parallel. Thus the pharmacology of V3/V1b receptors can not be transposed among different species.

scholarly journals Mannosylglycerate and Di-myo-Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress

2014 ◽  
Vol 80 (14) ◽  
pp. 4226-4233 ◽  
Author(s):  
Ana M. Esteves ◽  
Sanjeev K. Chandrayan ◽  
Patrick M. McTernan ◽  
Nuno Borges ◽  
Michael W. W. Adams ◽  
...  
Keyword(s):  
Heat Stress ◽  
Pyrococcus Furiosus ◽  
Inositol Phosphate ◽  
Compatible Solutes ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Cell Protection ◽  
Content Type ◽  
Molecular Events ◽  
Supraoptimal Temperatures

ABSTRACTMarine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities.Pyrococcus furiosusis a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed forP. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.

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