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 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.

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).

scholarly journals Multi-active Site Dynamics on a Molecular Cr/Co/Se Cluster Catalyst

2022 ◽  
Author(s):  
Jonathan Kephart ◽  
Benjamin Mitchell ◽  
Werner Kaminsky ◽  
Alexandra Velian
Keyword(s):  
Structural Analysis ◽  
Active Site ◽  
Resting State ◽  
Active Sites ◽  
Stepwise Mechanism ◽  
Nitrene Transfer ◽  
Catalytic Intermediates ◽  
Edge Site ◽  
Catalytically Active

This study provides detailed insights into the interconnected reactivity of the three catalytically active sites of an atomically precise nanocluster Cr3(py)3Co6Se8L6 (Cr3(py)3, L = Ph2PNTol–, Ph = phenyl, Tol = 4-tolyl). Catalytic and stoichiometric studies into tosyl azide activation and carbodiimide formation enabled the isolation and crystallographic characterization of key metal-nitrenoid catalytic intermediates, including the tris(nitrenoid) cluster Cr3(NTs)3, the catalytic resting state Cr3(NTs)3(CNtBu)3, and the mono(nitrenoid) cluster Cr3(NTs)(CNtBu)2. Nitrene transfer proceeds via a stepwise mechanism, with the three active sites engaging sequentially to produce carbodiimide. Comparative structural analysis and CNtBu bind-ing studies reveal that the chemical state of neighboring active sites regulates the affinity for substrates of an individual Cr-nitrenoid edge site, intertwining their reactivity through the inorganic support.

Acidic Functionalized Nanobohemite: An Active Catalyst for Methyl Ester Production

2019 ◽  
Vol 17 (11) ◽  
Author(s):  
Shokoufe Hosseini ◽  
G. R. Moradi ◽  
Kiumars Bahrami
Keyword(s):  
Active Sites ◽  
Free Acid ◽  
Reaction Medium ◽  
Biodiesel Production ◽  
Molar Ratio ◽  
Optimal Point ◽  
Acidic Catalysts ◽  
Boehmite Nanoparticles ◽  
Optimized Conditions

Abstract In the biodiesel production, acidic catalysts are ideally suitable for reacting with different oil sources at various free acid levels. On the other hand, the nanocatalysts can easily be propagated in the reaction medium and provide more accessible active sites for reaction. The aim of this work was to synthesize an acidic nanocatalyst based on boehmite nanoparticles then studying it to biodiesel production from soybean oil. Up to now, no reports were found on biodiesel production by this catalyst. After the synthesis and characterization of the catalyst, using response surface methodology (RSM), the optimized conditions for transesterification were 4.87 wt.% for catalyst dosage, 13:1 for the molar ratio of methanol to oil, 60 °C for reaction temperature, and 3 h for reaction time. At the optimal point, the production yield was 99.8 %. After six consecutive use of the catalyst, the yield dropped slightly (88 %). Consequently, the catalyst can be employed efficiently several runs in the production process.

scholarly journals Characterization of mTOR Activity and Metabolic Profile in Pediatric Rhabdomyosarcoma

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1947
Author(s):  
Luca Felkai ◽  
Ildikó Krencz ◽  
Dorottya Judit Kiss ◽  
Noémi Nagy ◽  
Gábor Petővári ◽  
...  
Keyword(s):  
Pentose Phosphate ◽  
Inhibitor Therapy ◽  
Metabolic Plasticity ◽  
Alternative Therapeutic Approach ◽  
Droplet Pcr ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Mtor Activity ◽  
Worse Prognosis ◽  
The Warburg Effect

mTOR activation has been observed in rhabdomyosarcoma (RMS); however, mTOR complex (mTORC) 1 inhibition has had limited success thus far. mTOR activation alters the metabolic pathways, which is linked to survival and metastasis. These pathways have not been thoroughly analyzed in RMSs. We performed immunohistochemistry on 65 samples to analyze the expression of mTOR complexes (pmTOR, pS6, Rictor), and several metabolic enzymes (phosphofructokinase, lactate dehydrogenase-A, β-F1-ATPase, glucose-6-phosphate dehydrogenase, glutaminase). RICTOR amplification, as a potential mechanism of Rictor overexpression, was analyzed by FISH and digital droplet PCR. In total, 64% of the studied primary samples showed mTOR activity with an mTORC2 dominance (82%). Chemotherapy did not cause any relevant change in mTOR activity. Elevated mTOR activity was associated with a worse prognosis in relapsed cases. RICTOR amplification was not confirmed in any of the cases. Our findings suggest the importance of the Warburg effect and the pentose-phosphate pathway beside a glutamine demand in RMS cells. The expression pattern of the studied mTOR markers can explain the inefficacy of mTORC1 inhibitor therapy. Therefore, we suggest performing a detailed investigation of the mTOR profile before administering mTORC1 inhibitor therapy. Furthermore, our findings highlight that targeting the metabolic plasticity could be an alternative therapeutic approach.

Molecular cloning and characterization of glucose-6-phosphate dehydrogenase from Brugia malayi

Parasitology ◽  
2013 ◽  
Vol 140 (7) ◽  
pp. 897-906 ◽  
Author(s):  
ANITA VERMA ◽  
MANISH K. SUTHAR ◽  
PAWAN K. DOHAREY ◽  
SMITA GUPTA ◽  
SUNITA YADAV ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Brugia Malayi ◽  
Pentose Phosphate ◽  
Subunit Molecular Weight ◽  
Optimum Ph ◽  
Sh Group ◽  
Glucose 6 Phosphate Dehydrogenase

SUMMARYGlucose-6-phosphate dehydrogenase (G6PD), a regulatory enzyme of the pentose phosphate pathway from Brugia malayi, was cloned, expressed and biochemically characterized. The Km values for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP) were 0·25 and 0·014 mm respectively. The rBmG6PD exhibited an optimum pH of 8·5 and temperature, 40 °C. Adenosine 5′ [γ-thio] triphosphate (ATP-γ-S), adenosine 5′ [β,γ-imido] triphosphate (ATP-β,γ-NH), adenosine 5′ [β-thio] diphosphate (ADP-β-S), Na+, K+, Li+ and Cu++ ions were found to be strong inhibitors of rBmG6PD. The rBmG6PD, a tetramer with subunit molecular weight of 75 kDa contains 0·02 mol of SH group per mol of monomer. Blocking the SH group with SH-inhibitors, led to activation of rBmG6PD activity by N-ethylmaleimide. CD analysis indicated that rBmG6PD is composed of 37% α-helices and 26% β-sheets. The unfolding equilibrium of rBmG6PD with GdmCl/urea showed the triphasic unfolding pattern along with the highly stable intermediate obtained by GdmCl.

TYPES OF ACTIVE CENTERS ON SURFACE OF PROMOTED IRON OXIDE CATALYST

2018 ◽  
Vol 61 (6) ◽  
pp. 61 ◽  
Author(s):  
Nikolai V. Dvoretskii ◽  
Lyubov G. Anikanova ◽  
Zoya G. Malysheva
Keyword(s):  
Iron Oxide ◽  
Active Sites ◽  
Oxide Catalyst ◽  
Coke Formation ◽  
Reaction Medium ◽  
Active Centers ◽  
Iron Oxide Catalyst ◽  
Oxygen Ion ◽  
Catalytically Active ◽  
Potassium Monoferrite

The phase and chemical composition of compounds in the potassium-iron-oxygen system in a wide range of molar ratios of potassium and iron was studied by X-ray phase analysis and atomic absorption spectroscopy. The catalytic properties and the mass fraction of coke deposits on ferritic systems of various composition are determined. It has been shown that at least two types of active sites are present on the surface of the iron oxide catalyst. The dehydrogenation centers include oxygen ion, ions of a promoting alkali metal, and ions of bivalent and triply charged iron, between which electron exchange takes place. Most probably such center is realized in the structure of potassium-polyferrite (K2Fe2+Fe3+10O17). The coke formation centers contain an unpromoted cluster consisting of oxygen ion and iron (III) ion, are realized in Fe3O4 and KFe11O17. Coke deposits on the surface of the catalyst block non-selective active sites and increase the selectivity of action. The probability of realization of clusters corresponding to the dehydrogenation centers is three orders of magnitude higher than in the places of phase contact, the aggregate of which contains the whole set of ions corresponding to dehydrogenation centers, for example, "magnetite + potassium monoferrite". The pure potassium β"-polyferrite provides an optimal concentration of selective centers on the surface of the catalyst, operates highly efficiently in the absence of negative external influences (catalyst re-recovery, corrosion reaction of the reaction medium, poisoning effect). Individual b²-polyferrites, like any catalytically active phase, are unstable, however, being in equilibrium with potassium monoferrate and magnetite, it is able to operate effectively for a long time and to withstand the negative effects of redox properties of the surrounding reaction medium. The presence of potassium monoferrite in the catalytically active system ensures the polyfunctionality of the action of contact, i.e. ability to self-regeneration. It is likely that in the structure of potassium monoferrite, centers for preventing coke formation and annealing of coke are realized, containing oxygen ion, iron ion, and alkaline promoter.Forcitation:Dvoretskii N.V., Anikanova L.G., Malysheva Z.G. Types of active centers on surface of promoted iron oxide catalyst. Izv. Vyssh. Uchebn. Zaved. Khim. Khim. Tekhnol. 2018. V. 61. N 6. P. 61-68

scholarly journals Biochemical Characterization and Structural Modeling of Fused Glucose-6-Phosphate Dehydrogenase-Phosphogluconolactonase from Giardia lamblia

2018 ◽  
Vol 19 (9) ◽  
pp. 2518 ◽  
Author(s):  
Laura Morales-Luna ◽  
Hugo Serrano-Posada ◽  
Abigail González-Valdez ◽  
Daniel Ortega-Cuellar ◽  
America Vanoye-Carlo ◽  
...  
Keyword(s):  
Giardia Lamblia ◽  
Tertiary Structure ◽  
Biochemical Characterization ◽  
Guanidine Hydrochloride ◽  
Pentose Phosphate ◽  
New Drugs ◽  
Oligomeric State ◽  
Functional Features ◽  
The Stability ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway and is highly relevant in the metabolism of Giardia lamblia. Previous reports suggested that the G6PD gene is fused with the 6-phosphogluconolactonase (6PGL) gene (6pgl). Therefore, in this work, we decided to characterize the fused G6PD-6PGL protein in Giardia lamblia. First, the gene of g6pd fused with the 6pgl gene (6gpd::6pgl) was isolated from trophozoites of Giardia lamblia and the corresponding G6PD::6PGL protein was overexpressed and purified in Escherichia coli. Then, we characterized the native oligomeric state of the G6PD::6PGL protein in solution and we found a catalytic dimer with an optimum pH of 8.75. Furthermore, we determined the steady-state kinetic parameters for the G6PD domain and measured the thermal stability of the protein in both the presence and absence of guanidine hydrochloride (Gdn-HCl) and observed that the G6PD::6PGL protein showed alterations in the stability, secondary structure, and tertiary structure in the presence of Gdn-HCl. Finally, computer modeling studies revealed unique structural and functional features, which clearly established the differences between G6PD::6PGL protein from G. lamblia and the human G6PD enzyme, proving that the model can be used for the design of new drugs with antigiardiasic activity. These results broaden the perspective for future studies of the function of the protein and its effect on the metabolism of this parasite as a potential pharmacological target.

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