scholarly journals Pathways leading to and from serine during growth of Pseudomonas AM1 in C1 compounds or succinate

1970 ◽  
Vol 117 (3) ◽  
pp. 563-572 ◽  
Author(s):  
J. Heptinstall ◽  
J. R. Quayle
Keyword(s):  
Dehydrogenase Activity ◽  
Reductase Activity ◽  
Methanol Dehydrogenase ◽  
Wild Type ◽  
Hydroxypyruvate Reductase ◽  
Phosphoglycerate Dehydrogenase ◽  
Specific Activities ◽  
Phenazine Methosulphate ◽  
Serine Biosynthesis ◽  
Enrichment Step

1. The following enzymes of the phosphorylated pathway of serine biosynthesis have been found in methanol- and succinate-grown Pseudomonas AM1: phosphoglycerate dehydrogenase, phosphoserine-α-oxoglutarate aminotransferase and phosphoserine phosphohydrolase. Their specific activities were similar in the organism grown on either substrate. 2. A procedure for preparation of auxotrophic mutants of Pseudomonas AM1 is described involving N-methyl-N′-nitro-N-nitrosoguanidine as mutagen and a penicillin enrichment step. 3. A mutant, M-15A, has been isolated that is unable to grow on methanol and that lacks phenazine methosulphate-linked methanol dehydrogenase. The mutant is able to grow on methylamine, showing that the amine is not oxidized by way of methanol. 4. Loss of methanol dehydrogenase activity in mutant M-15A led to loss of phenazine methosulphate-linked formaldehyde dehydrogenase activity showing that the same enzyme is probably responsible for both activities. 5. A mutant, 20B-L, has been isolated that cannot grow on any C1 compound tested but can grow on succinate. 6. Mutant 20B-L lacks hydroxypyruvate reductase, and revertants that regained the ability to grow on methanol, methylamine and formate contained hydroxypyruvate reductase activity at specific activities similar to that of the wild-type organism. This shows that hydroxypyruvate reductase is necessary for growth on methanol, methylamine and formate but not for growth on succinate. 7. The results suggest that during growth of Pseudomonas AM1 on C1 compounds, serine is converted into 3-phosphoglycerate by a non-phosphorylated pathway, whereas during growth on succinate, phosphoglycerate is converted into serine by a phosphorylated pathway.

scholarly journals The biosynthesis of serine and glycine in Pseudomonas AM1 with special reference to growth on carbon sources other than C1 compounds

1971 ◽  
Vol 121 (5) ◽  
pp. 753-762 ◽  
Author(s):  
W. Harder ◽  
J. R. Quayle
Keyword(s):  
Essential Role ◽  
Carbon Sources ◽  
Enzymic Activity ◽  
Serine Hydroxymethyltransferase ◽  
The Other ◽  
Wild Type ◽  
Phosphoserine Phosphatase ◽  
Wild Type Phenotype ◽  
Phosphoglycerate Dehydrogenase ◽  
Serine Biosynthesis

1. A mutant, 20S, of Pseudomonas AM1 was obtained that requires a supplement of serine to grow on succinate, lactate or ethanol. This mutant lacks phosphoserine phosphatase and revertants to wild-type phenotype regained this enzymic activity showing that the phosphorylated pathway of serine biosynthesis is necessary for growth on these three substrates. 2. The requirement for supplemental serine by mutant 20S could be met by glycine, suggesting that Pseudomonas AM1 can obtain C1 units from glycine. 3. Mutant 20S grows on C1 compounds at a lower rate compared with the wild type. Supplementation with serine stimulated the growth rate of the mutant suggesting that the phosphorylated pathway of serine biosynthesis plays some role, but not an essential role, during growth on C1 compounds. 4. A mutant, 82G, was obtained that requires a supplement of glycine to grow on succinate, lactate or ethanol. When grown in such supplemented media, the mutant lacks serine hydroxymethyltransferase and revertants to wild-type phenotype regained enzymic activity showing that during growth on succinate, lactate or ethanol, glycine is made from serine via serine hydroxymethyltransferase, and that the organism can obtain C1 units from glycine. 5. Mutant 82G grew on methanol and then contained serine hydroxymethyltransferase suggesting that this enzyme is necessary for growth on C1 compounds and that Pseudomonas AM1 may synthesize two such enzymes, one used in growth on C1 compounds, the other used in growth on other substrates. Mutant 82G might lack the latter enzyme. 6. Phosphoglycerate dehydrogenase is specifically inhibited by l-serine and the regulatory implications of this are discussed.

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.

'Liver-type' 11β-hydroxysteroid dehydrogenase cDNA encodes reductase but not dehydrogenase activity in intact mammalian COS-7 cells

1994 ◽  
Vol 13 (2) ◽  
pp. 167-174 ◽  
Author(s):  
S C Low ◽  
K E Chapman ◽  
C R W Edwards ◽  
J R Seckl
Keyword(s):  
Dehydrogenase Activity ◽  
Rat Liver ◽  
Mammalian Cells ◽  
Reductase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Intact Cells ◽  
Mmtv Ltr

ABSTRACT 11β-Hydroxysteroid dehydrogenase (11β-HSD) catalyses the metabolism of corticosterone to inert 11-dehydrocorticosterone, thus preventing glucocorticoid access to otherwise non-selective renal mineralocorticoid receptors (MRs), producing aldosterone selectivity in vivo. At least two isoforms of 11β-HSD exist. One isoform (11β-HSD1) has been purified from rat liver and an encoding cDNA cloned from a rat liver library. Transfection of rat 11β-HSD1 cDNA into amphibian cells with a mineralocorticoid phenotype encodes 11 β-reductase activity (activation of inert 11-dehydrocorticosterone) suggesting that 11β-HSD1 does not have the necessary properties to protect renal MRs from exposure to glucocorticoids. This function is likely to reside in a second 11β-HSD isoform. 11β-HSD1 is co-localized with glucocorticoid receptors (GRs) and may modulate glucocorticoid access to this receptor type. To examine the predominant direction of 11β-HSD1 activity in intact mammalian cells, and the possible role of 11β-HSD in regulating glucocorticoid access to GRs, we transfected rat 11β-HSD1 cDNA into a mammalian kidney-derived cell system (COS-7) which has little endogenous 11β-HSD activity or mRNA expression. Homogenates of COS-7 cells transfected with increasing amounts of 11β-HSD cDNA exhibited a dose-related increase in 11 β-dehydrogenase activity. In contrast, intact cells did not convert corticosterone to 11-dehydrocorticosterone over 24 h, but showed a clear dose-related 11β-reductase activity, apparent within 4 h of addition of 11-dehydrocorticosterone to the medium. To demonstrate that this reflected a change in functional intracellular glucocorticoids, COS-7 cells were co-transfected with an expression vector encoding GR and a glucocorticoid-inducible MMTV-LTR luciferase reporter construct, with or without 11β-HSD. Corticosterone induced MMTV-LTR luciferase expression in the presence or absence of 11β-HSD. 11-Dehydrocorticosterone was without activity in the absence of 11β-HSD, but induced MMTV-LTR luciferase activity in the presence of 11β-HSD. These results indicate that rat 11β-HSD1 can behave exclusively as a reductase in intact mammalian cells. Thus in some tissues in vivo, 11β-HSD1 may regulate ligand access to GRs by reactivating inert glucocorticoids.

Alterations in the components of ribonucleotide reductase in hydroxyurea-resistant hamster cells

1983 ◽  
Vol 3 (8) ◽  
pp. 741-748 ◽  
Author(s):  
Jim A. Wright ◽  
Joseph G. Cory
Keyword(s):  
Ribonucleotide Reductase ◽  
Reductase Activity ◽  
Antitumor Agent ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Wild Type ◽  
Type Analysis ◽  
Similar Mode ◽  
Ribonucleotide Reductase Activity ◽  
Effector Binding

Two components of mammalian ribonucleotide reductase have been separated by blue dextran-Sepharose chromatography from a hydroxyurea-resistant cell line, NcR-30A2, and its parental wild type. Analysis of reductase activity in these cells and the enzyme components reveals that there are three alterations involving ribonucleotide reductase activity in NcR-30A2 cells. There is an elevation in the effector-binding (EB) component, an elevation in the non-heine-ironcontaining (NHI) component, and an alteration in the NHI component that renders the enzyme less sensitive to inhibition by hydroxyurea. These findings easily account for the resistance of NcR-30A2 cells to the antitumor agent hydroxyurea, and to other drugs with a similar mode of action.

Amylase hyper-producing haploid recombinant strains of Thermomyces lanuginosus obtained by intraspecific protoplast fusion

2000 ◽  
Vol 46 (7) ◽  
pp. 669-673 ◽  
Author(s):  
K Rubinder ◽  
B S Chadha ◽  
S Singh ◽  
H S Saini
Keyword(s):  
Protoplast Fusion ◽  
Culture Filtrate ◽  
Catabolite Repression ◽  
Type Strain ◽  
Wild Type Strain ◽  
Thermomyces Lanuginosus ◽  
Wild Type ◽  
Haploid Strain ◽  
Recombinant Strains ◽  
Specific Activities

Amylase hyper-producing, catabolite-repression-resistant, recombinant strains were produced by intraspecific protoplast fusion of thermophilic fungus Thermomyces lanuginosus strains, using well-characterized, morphological, and 2-deoxy-D-glucose resistant markers. The fusant heterokaryons exhibited enhanced amylase activities as compared to the amylase hyper-producing parental strain (T2). Diploids derived from heterokaryons segregated to stable haploid recombinant strains. In the haploid strain (Tlh 4q), approximately 5-fold higher specific activities of α-amylase and glucoamylase in the culture filtrate were observed as compared to the wild-type strain (W0).Key words: Thermomyces lanuginosus, protoplast fusion, amylase hyper-producing strain, catabolite repression.

L-Serine regulates the activities of microglial cells that express very low level of 3-phosphoglycerate dehydrogenase, an enzyme forL-serine biosynthesis

2001 ◽  
Vol 64 (4) ◽  
pp. 392-401 ◽  
Author(s):  
Hiroki Sugishita ◽  
Yasuhide Kuwabara ◽  
Kazuko Toku ◽  
Lisa Doi ◽  
Lihua Yang ◽  
...  
Keyword(s):  
Microglial Cells ◽  
Low Level ◽  
Phosphoglycerate Dehydrogenase ◽  
Serine Biosynthesis

Separation of simian virus 40 large-T-antigen-transforming and origin-binding functions from the ability to block differentiation

1988 ◽  
Vol 8 (3) ◽  
pp. 1380-1384 ◽  
Author(s):  
V Cherington ◽  
M Brown ◽  
E Paucha ◽  
J St Louis ◽  
B M Spiegelman ◽  
...  
Keyword(s):  
Dehydrogenase Activity ◽  
Adipocyte Differentiation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Lipid Binding ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Glycerophosphate Dehydrogenase ◽  
Triglyceride Accumulation

Wild-type simian virus 40 large T antigen is very effective at blocking adipocyte differentiation in 3T3-F442A cells as assayed by triglyceride accumulation, induction of glycerophosphate dehydrogenase activity, and expression of mRNAs for glycerophosphate dehydrogenase, the adipocyte serine protease adipsin, and the putative lipid-binding protein adipocyte P2. Point mutants defective for either origin-specific DNA binding or transformation blocked differentiation as completely as wild type.

GLUCOSE AND RIBOSE OXIDATION BY PSEUDOMONAS FRAGI

1963 ◽  
Vol 41 (4) ◽  
pp. 1023-1034
Author(s):  
M. Leroux ◽  
H. L. A. Tarr
Keyword(s):  
Dehydrogenase Activity ◽  
Gluconic Acid ◽  
Soluble Fraction ◽  
Particulate Fraction ◽  
Pseudomonas Fragi ◽  
Dehydrogenase Enzyme ◽  
Specific Activities

The action of soluble and particulate fractions of sonically disrupted cells of Pseudomonas fragi on glucose and ribose was investigated. It was shown that ribose is oxidized by the particulate fraction to ribono-γ-lactone, but not further, thus verifying previous work. Evidence is presented in support of the fact that oxidation of both glucose and ribose is carried out by a single dehydrogenase enzyme, and that this enzyme is largely present in the particulate fraction. The soluble fraction possessed a lactonase enzyme which was purified slightly. This enzyme hydrolyzed glucono-δ-lactone but not glucono-γ-lactone. The washed particulate possessed no such lactonase activity. The soluble fraction possessed only about 4% of the dehydrogenase activity of the particulate fraction. Both the dehydrogenase and lactonase specific activities were similar when the organism was cultured in glucose or ribose-containing medium. Attempts to show that glucose was degraded by a phosphorolytic mechanism or that gluconic acid was degraded further failed. These findings are consistent with the fact that P. fragi oxidizes glucose to glucono-δ-lactone, and that this lactone is hydrolyzed to gluconic acid by a lactonase enzyme. No serious attempt was made to study the specificity of these enzymes, but it was observed that crude unwashed particulate fractions oxidized both galactose and mannose.

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