scholarly journals PnuT uses a facilitated diffusion mechanism for thiamine uptake

2017 ◽  
Vol 150 (1) ◽  
pp. 41-50 ◽  
Author(s):  
Michael Jaehme ◽  
Rajkumar Singh ◽  
Alisa A. Garaeva ◽  
Ria H. Duurkens ◽  
Dirk-Jan Slotboom
Keyword(s):  
Substrate Binding ◽  
Diffusion Mechanism ◽  
Vitamin B1 ◽  
Pyridine Nucleotide ◽  
Thiamine Pyrophosphate ◽  
Facilitated Diffusion ◽  
Nicotinamide Riboside ◽  
Thiamine Transport ◽  
Shewanella Woodyi ◽  
Thiamine Analogues

Membrane transporters of the bacterial pyridine nucleotide uptake (Pnu) family mediate the uptake of various B-type vitamins. For example, the PnuT transporters have specificity for vitamin B1 (thiamine). It has been hypothesized that Pnu transporters are facilitators that allow passive transport of the vitamin substrate across the membrane. Metabolic trapping by phosphorylation would then lead to accumulation of the transported substrates in the cytoplasm. However, experimental evidence for such a transport mechanism is lacking. Here, to determine the mechanism of thiamine transport, we purify PnuTSw from Shewanella woodyi and reconstitute it in liposomes to determine substrate binding and transport properties. We show that the electrochemical gradient of thiamine solely determines the direction of transport, consistent with a facilitated diffusion mechanism. Further, PnuTSw can bind and transport thiamine as well as the thiamine analogues pyrithiamine and oxythiamine, but does not recognize the phosphorylated derivatives thiamine monophosphate and thiamine pyrophosphate as substrates, consistent with a metabolic trapping mechanism. Guided by the crystal structure of the homologous nicotinamide riboside transporter PnuC, we perform mutagenesis experiments, which reveal residues involved in substrate binding and gating. The facilitated diffusion mechanism of transport used by PnuTSw contrasts sharply with the active transport mechanisms used by other bacterial thiamine transporters.

scholarly journals Real sequence effects on the search dynamics of transcription factors on DNA

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Maximilian Bauer ◽  
Emil S. Rasmussen ◽  
Michael A. Lomholt ◽  
Ralf Metzler
Keyword(s):  
Transcription Factors ◽  
Target Detection ◽  
Diffusion Mechanism ◽  
Coli Strain ◽  
Dna Looping ◽  
Real Sequence ◽  
Facilitated Diffusion ◽  
E Coli ◽  
Sliding Motion ◽  
Sequence Effects

Abstract Recent experiments show that transcription factors (TFs) indeed use the facilitated diffusion mechanism to locate their target sequences on DNA in living bacteria cells: TFs alternate between sliding motion along DNA and relocation events through the cytoplasm. From simulations and theoretical analysis we study the TF-sliding motion for a large section of the DNA-sequence of a common E. coli strain, based on the two-state TF-model with a fast-sliding search state and a recognition state enabling target detection. For the probability to detect the target before dissociating from DNA the TF-search times self-consistently depend heavily on whether or not an auxiliary operator (an accessible sequence similar to the main operator) is present in the genome section. Importantly, within our model the extent to which the interconversion rates between search and recognition states depend on the underlying nucleotide sequence is varied. A moderate dependence maximises the capability to distinguish between the main operator and similar sequences. Moreover, these auxiliary operators serve as starting points for DNA looping with the main operator, yielding a spectrum of target detection times spanning several orders of magnitude. Auxiliary operators are shown to act as funnels facilitating target detection by TFs.

Theoretical analysis of adenosine release from cardiac tissue as influenced by transport inhibitors

1983 ◽  
Vol 61 (8) ◽  
pp. 946-949 ◽  
Author(s):  
H. Wiener ◽  
W. G. Schützenberger ◽  
E. Tuisl ◽  
N. Kolassa
Keyword(s):  
Theoretical Analysis ◽  
Model Equation ◽  
Cardiac Tissue ◽  
Diffusion Mechanism ◽  
Facilitated Diffusion ◽  
Adenosine Release ◽  
Proposed Model ◽  
Transport Inhibitors ◽  
Transfer Term

The release of adenosine from cardiac tissue was simulated by use of a model equation which consists of a saturable transfer term for both unidirectional influx and efflux, representing a symmetrical facilitated diffusion mechanism. This proposed model can account for positive and negative changes in adenosine release from cardiac tissue brought about by competitive transport inhibitors.

Pyridine nucleotide metabolism by extracts derived from Haemophilus parasuis and H. pleuropneumoniae

1986 ◽  
Vol 32 (9) ◽  
pp. 733-737 ◽  
Author(s):  
T. O'Reilly ◽  
D. F. Niven
Keyword(s):  
Pyridine Nucleotide ◽  
Nucleotide Metabolism ◽  
Haemophilus Parasuis ◽  
Pyridine Nucleotides ◽  
Nicotinamide Riboside ◽  
Cell Fractions

A variety of biologically important pyridine nucleotides and precursors were examined for their capacities to serve as substrates for the synthesis of NAD by cell fractions derived from Haemophilus parasuis and H. pleuropneumoniae. Of the compounds tested, only NMN and nicotinamide riboside were converted to NAD. These reactions required ATP as co-substrate, and fractions from both organisms could also catalyze the ATP-dependent synthesis of NADP from NAD. In the absence of ATP, and depending on the pyridine compound under study, NAD, NMN, nicotinamide riboside, and also nicotinamide, were detected as products of catabolisra. It is concluded that these haemophili possess either three-membered pyridine nucleotide cycles or two-membered cycles with synthetic branches originating with nicotinamide riboside. It is also possible that the pyridine nucleotide cycles of both organisms have nonrecycling branches resulting in the "waste" of usable pyridine compound in the form of nicotinamide.

scholarly journals OR03-06 NAD+ Availability Modulates 11β-HSD1-Mediated Glucocorticoid Regeneration in Mouse Skeletal Muscle

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Yasir S Elhassan ◽  
Ali Kabli ◽  
Thomas Nielsen ◽  
Rachel Fletcher ◽  
Lucy Oakey ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Dehydrogenase Activity ◽  
Reductase Activity ◽  
Wild Type Mouse ◽  
Pyridine Nucleotide ◽  
Wild Type ◽  
Parent Molecule ◽  
Nicotinamide Riboside ◽  
Glucocorticoid Metabolism

Abstract 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is an NADPH-dependant reductase located in the sarcoplasmic reticulum (SR) lumen of skeletal muscle. It generates active glucocorticoids to regulate permissive and adaptive metabolism and contributes to the development of the Cushing’s syndrome phenotype in mice receiving oral corticosterone. The SR enzyme hexose-6-phosphate dehydrogenase (H6PDH) generates NADPH which supports 11β-HSD1 activity. H6PDH depletion disrupts the SR NADPH/NADP ratio leading 11β-HSD1 to assume glucocorticoid-inactivating dehydrogenase activity. Little is understood regarding routes to NAD(P)(H) biosynthesis and metabolism in the SR. Here we asked whether modulating cellular nicotinamide adenine dinucleotide (NAD+) availability (the parent molecule of NAD(P)(H)) would influence muscle 11β-HSD1 activity given its sensitivity to the SR NADPH/NADP ratio. We used FK866 to inhibit nicotinamide phospho-ribosyltransferase (NAMPT, rate-limiting enzyme in NAD+ biosynthesis) to deplete NAD(P)(H) in wild type mouse primary myotubes. FK866 treatment for 48h impaired cellular energetic status, reducing NAD+ (>90%), NADP+ (>50%) and ATP (>30%) without limiting cell viability. 11β-HSD1 reductase activity was decreased to 30% that of untreated cells (152±18 vs. 512±44 pmol/mg protein/h respectively, p<0.005). Employing H6PD knockout myotubes, NADP+-dependent 11β-HSD1 dehydrogenase activity was also impaired following NAMPT inhibition. The NAD+ precursor nicotinamide riboside (NR, 0.5mM), which bypasses NAMPT inhibition through the NR kinase pathway restored NAD+ levels and rapidly rescued 11β-HSD1 reductase activity in wild type and dehydrogenase activity in H6PD knockout myotubes. To assess this in vivo, we examined 11β-HSD1 reductase activity in muscle explants of inducible muscle-specific NAMPT knockout mice in which NAD+ levels are reduced by 90%, and show 40% lower activity compared to wild type explants (114±14 vs. 67±10 pmol/mg protein/h, p=0.04). These data suggest a novel level of redox-regulated 11β-HSD1-mediated glucocorticoid metabolism in skeletal muscle. These data also imply a pathway by which NAD+ status is communicated between the cytosol and the SR, which is contrary to the current belief that the pyridine nucleotide pool in these compartments is separate. NAMPT inhibition is being studied as a potential anti-cancer therapy and these data reveal hitherto unanticipated effects this therapy may have in a range of tissues.

The membrane permeability of nonelectrolytes and carbohydrate metabolism of Amazon fish red cells

1978 ◽  
Vol 56 (4) ◽  
pp. 863-869 ◽  
Author(s):  
Hyun Dju Kim ◽  
R. E. Isaacks
Keyword(s):  
Membrane Permeability ◽  
Carbohydrate Metabolism ◽  
Diffusion Mechanism ◽  
Red Cells ◽  
Facilitated Diffusion ◽  
Metabolic Substrates ◽  
Urea Permeability ◽  
Arapaima Gigas ◽  
Electric Eel ◽  
Order Of Magnitude

The membrane permeability to nonelectrolytes and carbohydrate metabolism were examined in red cells obtained from the Amazon fishes including the electric eel (Electrophorus electrocus), the arawana (Osteoglossum bicirrhosum), the pirarucu (Arapaima gigas), the lungfish (Lepidosireti paradoxa), and the armored catfish (Pterygoplichthys). Glucose permeability was fastest in the electric eel, followed by the lungfish. The red cells of the arawana were only slightly permeable to glucose. Both the armored catfish and the pirarucu red cells were found to be totally impermeable to glucose. There was no evidence for the presence of the facilitated diffusion mechanism for glucose transport in any of these fish red cells. In sharp contrast with glucose, red cells of all five species were quite permeable to ribose and urea. Urea permeability of red cells decreased in order of magnitude with the lungfish > the electric eel > the arawana > the armored catfish [Formula: see text] the pirarucu. The urea permeability of the lungfish was inhibited in the presence of phloretin.Of the two metabolic substrates, glucose but not ribose was metabolized to lactate with a concomitant contribution to ATP maintenance by the lungfish red cells. Even though the glucose-impervious pirarucu cells could not utilize glucose, ribose was readily metabolized by the pirarucu cells.

Two hydrate pseudopolymorphs of thiamine pyrophosphate: a dihydrate and a trihydrate

2013 ◽  
Vol 69 (7) ◽  
pp. 794-797
Author(s):  
Shu-Qiang Li ◽  
Ning-Hai Hu
Keyword(s):  
Hydrogen Bonds ◽  
Structural Study ◽  
Vitamin B1 ◽  
Pyrimidine Ring ◽  
Thiamine Pyrophosphate ◽  
Two Dimensional ◽  
Torsion Angles ◽  
Self Assembled ◽  
Methylene Group

Two hydrate pseudopolymorphs of 3-[(4-amino-2-methylpyrimidin-1-ium-5-yl)methyl]-4-methyl-1,3-thiazol-3-ium-5-yl hydrogen pyrophosphate (TPP),viz.a dihydrate, C12H18N4O7P2S·2H2O, (I), and a trihydrate, C12H18N4O7P2S·3H2O, (II), were obtained during a structural study of vitamin B1 coenzyme. In both compounds, TPP is a neutral zwitterion, with its pyrophosphate group doubly deprotonated and its pyrimidine ring protonated, and it assumes the usual `F' conformation in terms of the two torsion angles about the bonds by which the methylene group links the thiazolium and pyrimidinium rings [1.1 (3) and 79.7 (3)° for (I), and 2.0 (3) and 75.5 (3)° for (II)]. In (I), two TPP molecules are linked by a pair of O—H...O hydrogen bonds into a phosphate-pairing dimer. N—H...O hydrogen bonds connect the dimers into a sheet parallel to (101). In (II), the TPP molecules are self-assembled solely by N—H...O hydrogen bonds, generating a tape structure along [001]. A comparison of the four known hydrate pseudopolymorphs of TPP shows that the phosphate-pairing dimers are basic building units for the formation of two-dimensional networks.

scholarly journals Thiamine pyrophosphate riboswitches in Bacteroides species regulate transcription or translation of thiamine transport and biosynthesis genes

2019 ◽  
Author(s):  
Zachary A. Costliow ◽  
Patrick H. Degnan ◽  
Carin K. Vanderpool
Keyword(s):  
Gut Microbiome ◽  
Transcriptional Response ◽  
Thiamine Pyrophosphate ◽  
Individual Species ◽  
Vitamin B ◽  
Transcriptional Level ◽  
Thiamine Transport ◽  
Branched Chain ◽  
Species Specific ◽  
Thiamine Biosynthesis

AbstractThiamine (vitamin B1) and its phosphorylated precursors are necessary for decarboxylation reactions required in carbohydrate and branched chain amino acid metabolism. Due to its critical roles in central metabolism, thiamine is essential for human and animal hosts and their resident gut microbes. However, little is known about how thiamine availability shapes the composition of gut microbial communities and the physiology of individual species within those communities. Our previous work has implicated both thiamine biosynthesis and transport activities in the fitness of Bacteroides species. To better understand thiamine-dependent gene regulation in Bacteroides, we examined thiamine biosynthesis and transport genes in three representative species: Bacteroides thetaiotaomicron, Bacteroides uniformis, and Bacteroides vulgatus. All three species possess thiamine biosynthetic operons controlled by highly conserved cis-acting thiamine pyrophosphate (TPP) riboswitches. B. thetaiotaomicron and B. uniformis have additional TPP riboswitch-controlled operons encoding thiamine transport functions. Transcriptome analyses showed that each Bacteroides species had a distinct transcriptional response to exogenous thiamine. Analysis of transcript levels and translational fusions demonstrated that in B. thetaiotaomicron, the TPP riboswitch upstream of biosynthesis genes acts at the level of transcription, while TPP riboswitches upstream of transport operons work at the level of translation. In B. uniformis and B. vulgatus, TPP riboswitches work at the transcriptional level to control downstream operons. The varying responses to exogenous thiamine and use of varied regulatory mechanisms may play an important role in niche establishment by the Bacteroidetes in the complex and constantly shifting gut environment.ImportanceBacteroides species are important and abundant members of human gut microbiome communities. Their activities in the gut are influenced by constant changes in nutrient availability. In this study, we investigated the genetic basis of thiamine (Vitamin B1) uptake and biosynthesis in three representative Bacteroides species. We found species-specific differences in the response to exogenous thiamine, and distinct mechanisms for regulation of uptake and biosynthesis gene expression. Our work implies that gut Bacteroides have evolved distinct strategies for making or acquiring an essential nutrient. These mechanisms may play an important role in the success of Bacteroides in establishing a niche within complex gut microbiome communities.

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