scholarly journals Deuterium-labelled isotopomers of 2-C-methyl-d-erythritol as tools for the elucidation of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis

2000 ◽  
Vol 346 (3) ◽  
pp. 737-742 ◽  
Author(s):  
Lionel CHARON ◽  
Jean-François HOEFFLER ◽  
Catherine PALE-GROSDEMANGE ◽  
Luisa-Maria LOIS ◽  
Narciso CAMPOS ◽  
...  
Keyword(s):  
Deuterium Atom ◽  
Mep Pathway ◽  
Significant Activity ◽  
Hydrogen Atoms ◽  
Methyl Group ◽  
E Coli ◽  
Dimethylallyl Diphosphate ◽  
Metabolic Route ◽  
Starter Unit

Escherichia coli synthesizes its isoprenoids via the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The MC4100dxs::CAT strain, defective in deoxyxylulose-5-phosphate synthase, which is the first enzyme in this metabolic route, exclusively synthesizes its isoprenoids from exogenous 2-C-methyl-D-erythritol (ME) added to the culture medium. The fate of the hydrogen atoms in the MEP pathway was followed by the incorporation of [1,1-2H2]ME and [3,5,5,5-2H4]ME. The two C-1 hydrogen atoms of ME were found without any loss in the prenyl chain of menaquinone and/or ubiquinone on the carbon atoms derived from C-4 of isopentenyl diphosphate (IPP) and on the E-methyl group of dimethylallyl diphosphate (DMAPP), the C-5 hydrogen atoms on the methyl groups derived from IPP C-5 methyl group and the Z-methyl group of DMAPP. This showed that no changes in the oxidation state of these carbon atoms occurred in the reaction sequence between MEP and IPP. Furthermore, no deuterium scrambling was observed between the carbon atoms derived from C-4 and C-5 of IPP or DMAPP, suggesting a completely stereoselective IPP isomerase or no significant activity of this enzyme. The C-3 deuterium atom of [3,5,5,5-2H4]ME was preserved only in the DMAPP starter unit and was completely missing from all those derived from IPP. This finding, aided by the non-essential role of the IPP isomerase gene, suggests the presence in E. coli of two different routes towards IPP and DMAPP, starting from a common intermediate derived from MEP.

scholarly journals Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle

Plants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 462 ◽  
Author(s):  
Grégory Guirimand ◽  
Anthony Guihur ◽  
Catalina Perello ◽  
Michael Phillips ◽  
Samira Mahroug ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Cell Transformation ◽  
Mep Pathway ◽  
Protein Accumulation ◽  
Madagascar Periwinkle ◽  
Systematic Analysis ◽  
Metabolic Route ◽  
Genes Encoding ◽  
Young Leaves

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis.

Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis

2000 ◽  
Vol 353 (1) ◽  
pp. 59-67 ◽  
Author(s):  
Narciso CAMPOS ◽  
Manuel RODRÍGUEZ-CONCEPCIÓN ◽  
Susanna SAURET-GÜETO ◽  
Francesca GALLEGO ◽  
Luisa-María LOIS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Analysis ◽  
Mevalonate Pathway ◽  
Mep Pathway ◽  
Isopentenyl Diphosphate ◽  
Isopentenyl Diphosphate Isomerase ◽  
E Coli ◽  
Dimethylallyl Diphosphate ◽  
And Function ◽  
Synthetic Operon

Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) constitute the basic building block of isoprenoids, a family of compounds that is extraordinarily diverse in structure and function. IPP and DMAPP can be synthesized by two independent pathways: the mevalonate pathway and the recently discovered 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although the MEP pathway is essential in most eubacteria, algae and plants and has enormous biotechnological interest, only some of its steps have been determined. We devised a system suitable for the genetic analysis of the MEP pathway in Escherichia coli. A synthetic operon coding for yeast 5-diphosphomevalonate decarboxylase, human 5-phosphomevalonate kinase, yeast mevalonate kinase and E. coli isopentenyl diphosphate isomerase was incorporated in the chromosome of this bacterium. The expression of this operon allowed the synthesis of IPP and DMAPP from mevalonate added exogenously and complementation of lethal mutants of the MEP pathway. We used this system to show that the ygbP, ychB and ygbB genes are essential in E. coli and that the steps catalysed by the products of these genes belong to the trunk line of the MEP pathway.

scholarly journals Isoprenoid Biosynthesis in Synechocystis sp. Strain PCC6803 Is Stimulated by Compounds of the Pentose Phosphate Cycle but Not by Pyruvate or Deoxyxylulose-5-Phosphate

2002 ◽  
Vol 184 (18) ◽  
pp. 5045-5051 ◽  
Author(s):  
Yuri V. Ershov ◽  
R. Raymond Gantt ◽  
Francis X. Cunningham, ◽  
Elisabeth Gantt
Keyword(s):  
Pentose Phosphate ◽  
Isoprenoid Biosynthesis ◽  
Mep Pathway ◽  
Dihydroxyacetone Phosphate ◽  
E Coli ◽  
Dimethylallyl Diphosphate ◽  
Isomerase Activity ◽  
Pentose Phosphate Cycle ◽  
Initial Substrate ◽  
Alternative Routes

ABSTRACT The photosynthetic cyanobacterium Synechocystis sp. strain PCC6803 possesses homologs of known genes of the non-mevalonate 2-C-methyl-d-erythritol 2-phosphate (MEP) pathway for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Isoprenoid biosynthesis in extracts of this cyanobacterium, measured by incorporation of radiolabeled IPP, was not stimulated by pyruvate, an initial substrate of the MEP pathway in Escherichia coli, or by deoxyxylulose-5-phosphate, the first pathway intermediate in E. coli. However, high rates of IPP incorporation were obtained with addition of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P), as well as a variety of pentose phosphate cycle compounds. Fosmidomycin (at 1 μM and 1 mM), an inhibitor of deoxyxylulose-5-phosphate reductoisomerase, did not significantly inhibit phototrophic growth of the cyanobacterium, nor did it affect [14C]IPP incorporation stimulated by DHAP plus GA3P. To date, it has not been possible to unequivocally demonstrate IPP isomerase activity in this cyanobacterium. The combined results suggest that the MEP pathway, as described for E. coli, is not the primary path by which isoprenoids are synthesized under photosynthetic conditions in Synechocystis sp. strain PCC6803. Our data support alternative routes of entry of pentose phosphate cycle substrates derived from photosynthesis.

scholarly journals The role of a cholesta-8,14-dien-3β-ol system in cholesterol biosynthesis

1969 ◽  
Vol 111 (5) ◽  
pp. 757-761 ◽  
Author(s):  
M. Akhtar ◽  
I. A. Watkinson ◽  
A. D. Rahimtula ◽  
D. C. Wilton ◽  
K. A. Munday
Keyword(s):  
Hydrogen Atom ◽  
Cholesterol Biosynthesis ◽  
Tritiated Water ◽  
Mevalonic Acid ◽  
Hydrogen Atoms ◽  
Steroid Biosynthesis ◽  
Methyl Group

The biosynthesis of cholesterol from squalene and tritiated water is described. Degradation of the cholesterol indicated that C-15 may be involved in cholesterol biosynthesis. In accordance with this view it is shown that in the conversion of [2RS−3H2]mevalonic acid into cholesterol one of the hydrogen atoms at C-15 is removed. A mechanism for the removal of the 14α-methyl group in steroid biosynthesis that involves the labilization of a C-15 hydrogen atom is outlined. In accordance with the requirement of this scheme it is shown that 4,4′-dimethyl-cholesta-8,14-dien-3β-ol is converted into cholesterol.

The Local Sanarelli-Shwartzman Phenomenon in Rats Induced by Human Leukaemic Cells

1973 ◽  
Vol 29 (02) ◽  
pp. 353-362
Author(s):  
J Lisiewicz ◽  
A Pituch ◽  
J. A Litwin
Keyword(s):  
Chronic Lymphocytic Leukaemia ◽  
Intravenous Injection ◽  
Peripheral Blood ◽  
Lymphocytic Leukaemia ◽  
Acute Myeloblastic Leukaemia ◽  
Demarcation Line ◽  
E Coli ◽  
Leukaemic Cells ◽  
Shwartzman Phenomenon

SummaryThe local Sanarelli-Shwartzman phenomenon (SSP-L) in the skin of 30 rats was induced by an intr a cutaneous sensitizing injection of leukaemic leucocytes isolated from the peripheral blood of patients with chronic lymphocytic leukaemia (CLL), acute myeloblastic leukaemia (AL) and chronic granulocytic leukaemia (CGL) and challenged by an intravenous injection of 100(μ of E. coli endotoxin. SSP-L was observed in 7 rats after injection of CLL lymphocytes and in 6 and 2 rats after AL myeloblasts and the CGL granulocytes, respectively. The lesions in the skin after AL myeloblasts appeared in a shorter time and were of longer duration compared with those observed after CLL lymphocytes and CGL granulocytes. Histologically, the lesions consisted of areas of destruction in the superficial layers of the skin ; the demarcation line showed the presence of neutrophils, macrophages and erythrocytes. Haemorrhages and fibrin deposits near the demarcation line were larger after injection of CLL lymphocytes and AL myeloblasts than after CGL granulocytes. The possible role of leucocyte procoagulative substances in the differences observed have been discussed.

Bioenhancing Effect of Cow Urine Distillate and Pepper Extract on Antibacterial Activity of Azadirachta Indica Leaves

1970 ◽  
Vol 9 (2) ◽  
pp. 3212-3214
Author(s):  
Pramod Dhakal ◽  
Ankit a Achary ◽  
Vedamurthy Joshi
Keyword(s):  
Antibacterial Activity ◽  
Azadirachta Indica ◽  
Pathogenic Bacteria ◽  
Ethanol Extract ◽  
Watery Diarrhea ◽  
Diffusion Method ◽  
E Coli ◽  
Drug Molecules ◽  
Cow Urine

Bioenhancers are drug facilitator which do not show the typical drug activity but in combination to enhance the activity of other molecule in several way including increase the bioavailability of drug across the membrane, potentiating the drug molecules by conformational interaction, acting as receptor for drug molecules and making target cell more receptive to drugs and promote and increase the bioactivity or bioavailability or the uptake of drugs in combination therapy. The objective of the present study was to evaluate the antibacterial and activity of combination in Azadirachta indica extract with cow urine distillate and pepper extract against common pathogenic bacteria, a causative agent of watery diarrhea. It has been found that Indian indigenous cow urine and its distillate also possess bioenhancing ability. Bioenhancing role of cow urine distillate (CUD) and pepper extract was investigated on antibacterial activity of ethanol extract of Azadirachta indica. Antibacterial activity of ethanol extract neem alone and in combination with CUD and pepper extract were determined the ATCC strains against Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and E-coli by cup plate diffusion method. Ethanol extract of neem has showed more effect on P. aeruginosa, E-coli than S. aureus and K. pneumonia with combination of CUD and pepper extract. CUD and pepper did not show any inhibition of test bacteria in low concentration. The antibacterial effect of combination of extract and CUD was higher than the inhibition caused by extract alone and is suggestive of the bioenhancing role of cow urine distillate and pepper. Moreover, inhibition of test bacteria was observed with less concentration of extract on combining with CUD

scholarly journals E. coli Enterotoxin LtB Enhances Vaccine-Induced Anti-H. pylori Protection by Promoting Leukocyte Migration into Gastric Mucus via Inflammatory Lesions

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 982
Author(s):  
Xiaoyan Peng ◽  
Rongguang Zhang ◽  
Chen Wang ◽  
Feiyan Yu ◽  
Mingyang Yu ◽  
...  
Keyword(s):  
Cellular Immunity ◽  
Protective Efficacy ◽  
Gastric Injury ◽  
Oral Vaccines ◽  
Gastric Mucus ◽  
E Coli ◽  
Non Invasive ◽  
Considerable Impact ◽  
H Pylori

Current studies indicate that the anti-H. pylori protective efficacy of oral vaccines to a large extent depends on using mucosal adjuvants like E. coli heat-lable enterotoxin B unit (LtB). However, the mechanism by which Th17/Th1-driven cellular immunity kills H. pylori and the role of LtB remains unclear. Here, two L. lactis strains, expressing H. pylori NapA and LtB, respectively, were orally administrated to mice. As observed, the administration of LtB significantly enhanced the fecal SIgA level and decreased gastric H. pylori colonization, but also markedly aggravated gastric inflammatory injury. Both NapA group and NapA+LtB group had elevated splenocyte production of IL-8, IL-10, IL-12, IL-17, IL-23 and INF-γ. Notably, gastric leukocytes’ migration or leakage into the mucus was observed more frequently in NapA+LtB group than in NapA group. This report is the first that discusses how LtB enhances vaccine-induced anti-H. pylori efficacy by aggravating gastric injury and leukocytes’ movement into the mucus layer. Significantly, it brings up a novel explanation for the mechanism underlying mucosal cellular immunity destroying the non-invasive pathogens. More importantly, the findings suggest the necessity to further evaluate LtB’s potential hazards to humans before extending its applications. Thus, this report can provide considerable impact on the fields of mucosal immunology and vaccinology.

scholarly journals Reconstitution in Proteoliposomes of the Recombinant Human Riboflavin Transporter 2 (SLC52A2) Overexpressed in E. coli

2019 ◽  
Vol 20 (18) ◽  
pp. 4416 ◽  
Author(s):  
Lara Console ◽  
Maria Tolomeo ◽  
Matilde Colella ◽  
Maria Barile ◽  
Cesare Indiveri
Keyword(s):  
Cardiovascular Disease ◽  
Amino Acids ◽  
Risk Factor ◽  
Biologically Active ◽  
Suitable Model ◽  
Native Protein ◽  
E Coli ◽  
Few Data ◽  
Riboflavin Transport

Background: the SLC52A2 gene encodes for the riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed. It mediates the transport of Riboflavin across cell membranes. Riboflavin plays a crucial role in cells since its biologically active forms, FMN and FAD, are essential for the metabolism of carbohydrates, amino acids, and lipids. Mutation of the Riboflavin transporters is a risk factor for anemia, cancer, cardiovascular disease, neurodegeneration. Inborn mutations of SLC52A2 are associated with Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset. In spite of the important metabolic and physio/pathological role of this transporter few data are available on its function and regulation. Methods: the human recombinant RFVT2 has been overexpressed in E. coli, purified and reconstituted into proteoliposomes in order to characterize its activity following the [3H]Riboflavin transport. Results: the recombinant hRFVT2 showed a Km of 0.26 ± 0.07 µM and was inhibited by lumiflavin, FMN and Mg2+. The Riboflavin uptake was also regulated by Ca2+. The native protein extracted from fibroblast and reconstituted in proteoliposomes also showed inhibition by FMN and lumiflavin. Conclusions: proteoliposomes represent a suitable model to assay the RFVT2 function. It will be useful for screening the mutation of RFVT2.

scholarly journals The essential role of mRNA degradation in understanding and engineering E. coli metabolism

2021 ◽  
pp. 107805
Author(s):  
Charlotte Roux ◽  
Thibault Etienne ◽  
Eliane Hajnsdorf ◽  
Delphine Ropers ◽  
A.J. Carpousis ◽  
...  
Keyword(s):  
Essential Role ◽  
Mrna Degradation ◽  
E Coli
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