scholarly journals Glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase: a unique bifunctional enzyme from Plasmodium falciparum

2011 ◽  
Vol 436 (3) ◽  
pp. 641-650 ◽  
Author(s):  
Esther Jortzik ◽  
Boniface M. Mailu ◽  
Janina Preuss ◽  
Marina Fischer ◽  
Lars Bode ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Pentose Phosphate Pathway ◽  
Drug Target ◽  
Pentose Phosphate ◽  
Bifunctional Enzyme ◽  
Functional Differences ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
First Time ◽  
Human Rbcs

The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.

scholarly journals Glucose-6-Phosphate Dehydrogenase::6-Phosphogluconolactonase from the Parasite Giardia lamblia. A Molecular and Biochemical Perspective of a Fused Enzyme

2021 ◽  
Vol 9 (8) ◽  
pp. 1678
Author(s):  
Laura Morales-Luna ◽  
Abigail González-Valdez ◽  
Beatriz Hernández-Ochoa ◽  
Roberto Arreguin-Espinosa ◽  
Daniel Ortega-Cuellar ◽  
...  
Keyword(s):  
Giardia Lamblia ◽  
Drug Target ◽  
Active Sites ◽  
Reaction Medium ◽  
Structural Difference ◽  
Pentose Phosphate ◽  
Catalytically Active ◽  
The Individual ◽  
Glucose 6 Phosphate Dehydrogenase

Giardia lamblia is a single-celled eukaryotic parasite with a small genome and is considered an early divergent eukaryote. The pentose phosphate pathway (PPP) plays an essential role in the oxidative stress defense of the parasite and the production of ribose-5-phosphate. In this parasite, the glucose-6-phosphate dehydrogenase (G6PD) is fused with the 6-phosphogluconolactonase (6PGL) enzyme, generating the enzyme named G6PD::6PGL that catalyzes the first two steps of the PPP. Here, we report that the G6PD::6PGL is a bifunctional enzyme with two catalytically active sites. We performed the kinetic characterization of both domains in the fused G6PD::6PGL enzyme, as well as the individual cloned G6PD. The results suggest that the catalytic activity of G6PD and 6PGL domains in the G6PD::6PGL enzyme are more efficient than the individual proteins. Additionally, using enzymatic and mass spectrometry assays, we found that the final metabolites of the catalytic reaction of the G6PD::6PGL are 6-phosphoglucono-δ-lactone and 6-phosphogluconate. Finally, we propose the reaction mechanism in which the G6PD domain performs the catalysis, releasing 6-phosphoglucono-δ-lactone to the reaction medium. Then, this metabolite binds to the 6PGL domain catalyzing the hydrolysis reaction and generating 6-phosphogluconate. The structural difference between the G. lamblia fused enzyme G6PD::6PGL with the human G6PD indicate that the G6PD::6PGL is a potential drug target for the rational synthesis of novels anti-Giardia drugs.

scholarly journals Characterization of the terminal stages of chlorophyll (ide) synthesis in etioplast membrane preparations

1975 ◽  
Vol 152 (3) ◽  
pp. 623-655 ◽  
Author(s):  
W T Griffiths
Keyword(s):  
Pentose Phosphate Pathway ◽  
Chlorophyll Biosynthesis ◽  
Pentose Phosphate ◽  
Activation Process ◽  
Active Complex ◽  
Sh Reagents ◽  
Intermittent Illumination ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Etioplast Membranes

1. Chlorophyll (ide) formation from protochlorophyll (ide) that is normally inactive was demonstrated in etioplast membranes isolated from maize and barlley plants, the process being dependent on intermittent illumination and the addition of NADPH. 2. The addition of NADPH to the membranes was shown to result in the conversion of inactive protochlorophyll (ide) absorbing at about 630 nm into a form(s) with light-absorption maxima at about 640 and 652 nm, both of which disappear when chlorophyll (ide) is formed on illumination. 3. The temperature-dependence of the activation process and its response to a variety of reagents were examined. From these, the conclusion is drawn that -SH groups are involved in the activation but in the active complex these are unavailable for reaction with -SH reagents. 4. Evidence is presented for the occurrence of glucose 6-phosphate dehydrogenase activity within etioplasts and the suggestion is made that the oxidative pentose phosphate pathway can provide the NADPH required for chlorophyll biosynthesis during the early stages of greening.

scholarly journals CBMT-08. IN VIVO EVALUATION OF PENTOSE PHOSPHATE PATHWAY ACTIVITY IN ORTHOTOPIC GLIOMA USING HYPERPOLARIZED δ-[1-13C]GLUCONOLACTONE

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi34-vi34
Author(s):  
Georgios Batsios ◽  
Pavithra Viswanath ◽  
Peng Cao ◽  
Celine Taglang ◽  
Elavarasan Subramani ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Tumor Development ◽  
Pentose Phosphate ◽  
Response To Therapy ◽  
Metabolic Imaging ◽  
Normal Brain ◽  
In Vivo Evaluation ◽  
First Time ◽  
Orthotopic Glioma

Abstract The pentose phosphate pathway (PPP) generates NADPH and ribose 5-phosphate, which are involved in the scavenging of reactive oxygen species and the synthesis of nucleotides. As such, the PPP is typically upregulated in cancer cells to address the metabolic needs of rapid cell proliferation. Imaging PPP upregulation could therefore be useful in tumor assessment. One intermediate of the pathway is 6-phospho-δ-gluconolactone (6P-δ-GL), which is produced by phosphorylation of δ-gluconolactone. 6P-δ-GL is further metabolized to 6-phospho-gluconate (6PG). The goal of our study was to evaluate, for the first time, whether hyperpolarized (HP) δ-[1-13C]gluconolactone can be used to assess PPP flux and detect the presence of tumor in an orthotopic glioma rat model. Athymic nude rats bearing orthotropic U87 tumors or age-matched tumor-free controls were investigated. HP studies were performed following intravenous injection of HP δ-[1-13C]gluconolactone and metabolic images using a flyback spectral-spatial echo-planar spectroscopic imaging pulse were acquired. The data were processed using in-house Matlab code. 6P-δ-GL and 6-phospho-γ-[1-13C]gluconolactone were observed in all rats ~10 seconds after HP δ-[1-13C]gluconolactone injection, followed ~5 seconds later by production of 6PG observed at 179.3ppm. These data indicate that HP δ-[1-13C]gluconolactone likely crosses the blood-brain barrier, consistent with its transport via glucose transporters, and is rapidly metabolized. Importantly, 6PG was significantly higher in tumor voxels. The ratio of 6PG-to-6P-δ-GL was comparable in normal brain and in normal-appearing contralateral brain of tumor-bearing rats at 0.43±0.09 and 0.45±0.06 respectively (p=0.85), but significant higher in the tumor regions at 0.70±0.11 (p=0.04 and p=0.02 respectively), consistent with the elevated PPP flux that typically occurs in tumor cells. Our results indicate, to our knowledge for the first time, that metabolism of HP δ-[1-13C]gluconolactone can be assessed in the brain and that elevated 6PG production in glioma provides a potential metabolic imaging approach to probe tumor development, recurrence and response to therapy.

scholarly journals Molecular Characterization of the First Two Enzymes of the Pentose-phosphate Pathway of Trypanosoma brucei

2000 ◽  
Vol 275 (36) ◽  
pp. 27559-27565 ◽  
Author(s):  
Francis Duffieux ◽  
Joris Van Roy ◽  
Paul A.M. Michels ◽  
Fred R. Opperdoes
Keyword(s):  
Molecular Characterization ◽  
Trypanosoma Brucei ◽  
Pentose Phosphate Pathway ◽  
Pentose Phosphate

Identification and Characterization of Novel Human Glucose-6-Phosphate Dehydrogenase Inhibitors

2012 ◽  
Vol 18 (3) ◽  
pp. 286-297 ◽  
Author(s):  
Janina Preuss ◽  
Adam D. Richardson ◽  
Anthony Pinkerton ◽  
Michael Hedrick ◽  
Eduard Sergienko ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Basic Research ◽  
Pentose Phosphate ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Key Enzyme ◽  
Small Molecule Compound ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Identification And Characterization

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone with parallel reduction of NADP+. Several human diseases, including cancer, are associated with increased G6PD activity. To date, only a few G6PD inhibitors have been available. However, adverse side effects and high IC50 values hamper their use as therapeutics and basic research probes. In this study, we developed a high-throughput screening assay to identify novel human G6PD (hG6PD) inhibitors. Screening the LOPAC (Sigma-Aldrich; 1280 compounds), Spectrum (Microsource Discovery System; 1969 compounds), and DIVERSet (ChemBridge; 49 971 compounds) small-molecule compound collections revealed 139 compounds that presented ≥50% hG6PD inhibition. Hit compounds were further included in a secondary and orthogonal assay in order to identify false-positives and to determine IC50 values. The most potent hG6PD inhibitors presented IC50 values of <4 µM. Compared with the known hG6PD inhibitors dehydroepiandrosterone and 6-aminonicotinamide, the inhibitors identified in this study were 100- to 1000-fold more potent and showed different mechanisms of enzyme inhibition. One of the newly identified hG6PD inhibitors reduced viability of the mammary carcinoma cell line MCF10-AT1 (IC50 ~25 µM) more strongly than that of normal MCF10-A cells (IC50 >50 µM).

Respiratory enzyme activities during germination inBrassicaseed lots of differing vigour

1996 ◽  
Vol 6 (4) ◽  
pp. 165-174 ◽  
Author(s):  
Mary Bettey ◽  
W.E. Finch-Savage
Keyword(s):  
Oxygen Consumption ◽  
Pentose Phosphate Pathway ◽  
Sharp Increase ◽  
Artificial Aging ◽  
Pentose Phosphate ◽  
Untreated Seed ◽  
Seed Vigour ◽  
Regulatory Enzymes ◽  
Rate Of Oxygen Consumption ◽  
Glucose 6 Phosphate Dehydrogenase

AbstractThe rate of oxygen consumption by cabbage seeds increased on imbibition and there was a further sharp increase on germination. This was delayed in artificially aged seeds of low vigour. The increases in oxygen consumption reflect the increased oxidation of carbohydrates via respiratory pathways. The activities of key regulatory enzymes of glycolysis and the oxidative pentose phosphate pathway were measured in aged and unaged seed lots of cabbage. Differences in the activities of glucose 6-phosphate dehydrogenase and pyrophosphate:fructose 6-phosphate 1-phosphotransferase were correlated with the rate of germination (T50) in seed lots with large differences in seed vigour induced experimentally by artificial aging. However, the activities of these enzymes could not be used to distinguish between untreated seed lots which had smaller vigour differences apparent only under stress. The enzymes are presumably not controlling and determining seed vigour, but merely reflecting actual seed performance under the current conditions.

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