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.

scholarly journals Identification of the NADP+ Structural Binding Site and Coenzyme Effect on the Fused G6PD::6PGL Protein from Giardia lamblia

Biomolecules ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 46
Author(s):  
Laura Morales-Luna ◽  
Abigail González-Valdez ◽  
Yudibeth Sixto-López ◽  
José Correa-Basurto ◽  
Beatriz Hernández-Ochoa ◽  
...  
Keyword(s):  
Binding Site ◽  
Giardia Lamblia ◽  
Md Simulation ◽  
Thermal Inactivation ◽  
Homo Sapiens ◽  
3D Structure ◽  
Protozoan Parasite ◽  
Pentose Phosphate ◽  
The Stability ◽  
Glucose 6 Phosphate Dehydrogenase

Giardia lambia is a flagellated protozoan parasite that lives in the small intestine and is the causal agent of giardiasis. It has been reported that G. lamblia exhibits glucose-6-phosphate dehydrogenase (G6PD), the first enzyme in the pentose phosphate pathway (PPP). Our group work demonstrated that the g6pd and 6pgl genes are present in the open frame that gives rise to the fused G6PD::6PGL protein; where the G6PD region is similar to the 3D structure of G6PD in Homo sapiens. The objective of the present work was to show the presence of the structural NADP+ binding site on the fused G6PD::6PGL protein and evaluate the effect of the NADP+ molecule on protein stability using biochemical and computational analysis. A protective effect was observed on the thermal inactivation, thermal stability, and trypsin digestions assays when the protein was incubated with NADP+. By molecular docking, we determined the possible structural-NADP+ binding site, which is located between the Rossmann fold of G6PD and 6PGL. Finally, molecular dynamic (MD) simulation was used to test the stability of this complex; it was determined that the presence of both NADP+ structural and cofactor increased the stability of the enzyme, which is in agreement with our experimental results.

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.

Signal transducer gp130: biochemical characterization of the three membrane-proximal extracellular domains and evaluation of their oligomerization potential

2001 ◽  
Vol 356 (2) ◽  
pp. 605-612 ◽  
Author(s):  
Stefan PFLANZ ◽  
Thomas KERNEBECK ◽  
Bernd GIESE ◽  
Andreas HERRMANN ◽  
Michael PACHTA-NICK ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Biochemical Characterization ◽  
Transmembrane Protein ◽  
Proximal Part ◽  
Cd Spectroscopy ◽  
Size Exclusion ◽  
Type I ◽  
Proximal Half ◽  
Functional Features

Glycoprotein 130 (gp130) is a type I transmembrane protein and serves as the common signal-transducing receptor subunit of the interleukin-6-type cytokines. Whereas the membrane-distal half of the gp130 extracellular part confers ligand binding and has been subject to intense investigation, the structural and functional features of its membrane-proximal half are poorly understood. On the basis of predictions of tertiary structure, the membrane-proximal part consists of three fibronectin-type-III-like domains D4, D5 and D6. Here we describe the bacterial expression of the polypeptides predicted to comprise each of these three domains. The recombinant proteins were refolded from solubilized inclusion bodies in vitro, purified to homogeneity and characterized by means of size-exclusion chromatography and CD spectroscopy. For the first time the prediction of three individual membrane-proximal protein domains for gp130has been verified experimentally. The three domains do not show intermediate-affinity or high-affinity interactions between each other. Mapping of a neutralizing gp130 monoclonal antibody against D4 suggested a particular functional role of this domain for gp130 activation, because above that an intrinsic tendency for low-affinity oligomerization was demonstrated for D4.

scholarly journals Discovery of New Uncompetitive Inhibitors of Glucose-6-Phosphate Dehydrogenase

2014 ◽  
Vol 19 (10) ◽  
pp. 1362-1371 ◽  
Author(s):  
Gustavo F. Mercaldi ◽  
Americo T. Ranzani ◽  
Artur T. Cordeiro
Keyword(s):  
Side Effects ◽  
Blood Cells ◽  
Structural Features ◽  
Pentose Phosphate ◽  
New Drugs ◽  
Biosynthetic Pathways ◽  
Human Homologue ◽  
Structure Activity ◽  
Quinazolinone Derivatives ◽  
Glucose 6 Phosphate Dehydrogenase

The enzyme glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the oxidative branch of the pentose phosphate pathway, which provides cells with NADPH, an essential cofactor for many biosynthetic pathways and antioxidizing enzymes. In Trypanosoma cruzi, the G6PDH has being pursued as a relevant target for the development of new drugs against Chagas disease. At present, the best characterized inhibitors of T. cruzi G6PDH are steroidal halogenated compounds derivatives from the mammalian hormone precursor dehydroepiandrosterone, which indeed are also good inhibitors of the human homologue enzyme. The lack of target selectivity might result in hemolytic side effects due to partial inhibition of human G6PDH in red blood cells. Moreover, the treatment of Chagas patients with steroidal drugs might also cause undesired androgenic side effects. Aiming to identify of new chemical classes of T. cruzi G6PDH inhibitors, we performed a target-based high-throughput screen campaign against a commercial library of diverse compounds. Novel TcG6PDH inhibitors were identified among thienopyrimidine and quinazolinone derivatives. Preliminary structure activity relationships for the identified hits are presented, including structural features that contribute for selectivity toward the parasite enzyme. Our results indicate that quinazolinones are promising hits that should be considered for further optimization.

scholarly journals Cloning, Expression, and Characterization of a Psychrophilic Glucose 6-Phosphate Dehydrogenase from Sphingomonas sp. PAMC 26621

2019 ◽  
Vol 20 (6) ◽  
pp. 1362 ◽  
Author(s):  
Kiet TranNgoc ◽  
Nhung Pham ◽  
ChangWoo Lee ◽  
Sei-Heon Jang
Keyword(s):  
Escherichia Coli ◽  
Secondary Structure ◽  
Tertiary Structure ◽  
Activation Enthalpy ◽  
Pentose Phosphate ◽  
Flexible Structure ◽  
Psychrophilic Bacterium ◽  
Km Value ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.363) is a crucial regulatory enzyme in the oxidative pentose phosphate pathway that provides reductive potential in the form of NADPH, as well as carbon skeletons for the synthesis of macromolecules. In this study, we report the cloning, expression, and characterization of G6PD (SpG6PD1) from a lichen-associated psychrophilic bacterium Sphingomonas sp. PAMC 26621. SpG6PD1 was expressed in Escherichia coli as a soluble protein, having optimum activity at pH 7.5–8.5 and 30 °C for NADP+ and 20 °C for NAD+. SpG6PD1 utilized both NADP+ and NAD+, with the preferential utilization of NADP+. A high Km value for glucose 6-phosphate and low activation enthalpy (ΔH‡) compared with the values of mesophilic counterparts indicate the psychrophilic nature of SpG6PD1. Despite the secondary structure of SpG6PD1 being maintained between 4–40 °C, its activity and tertiary structure were better preserved between 4–20 °C. The results of this study indicate that the SpG6PD1 that has a flexible structure is most suited to a psychrophilic bacterium that is adapted to a permanently cold habitat.

NADPH Producing Enzymes as Promising Drug Targets for Chagas Disease

2019 ◽  
Vol 26 (36) ◽  
pp. 6564-6571
Author(s):  
Artur T. Cordeiro
Keyword(s):  
Drug Target ◽  
Drug Targets ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Acid Synthesis ◽  
Pentose Phosphate ◽  
New Drugs ◽  
Trypanosomatid Parasites ◽  
Drug Target Validation ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Glucose 6 Phosphate Dehydrogenase

Reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) is a cofactor used in different anabolic reactions, such as lipid and nucleic acid synthesis, and for oxidative stress defense. NADPH is essential for parasite growth and viability. In trypanosomatid parasites, NADPH is supplied by the oxidative branch of the pentose phosphate pathway and by enzymes associated with the citric acid cycle. The present article will review recent achievements that suggest glucose-6-phosphate dehydrogenase and the cytosolic isoform of the malic enzyme as promising drug targets for the discovery of new drugs against Trypanosoma cruzi and T. brucei. Topics involving an alternative strategy in accelerating T. cruzi drug-target validation and the concept of drug-target classification will also be revisited.

scholarly journals Metabolic Anti-Cancer Effects of Melatonin: Clinically Relevant Prospects

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3018
Author(s):  
Marek Samec ◽  
Alena Liskova ◽  
Lenka Koklesova ◽  
Kevin Zhai ◽  
Elizabeth Varghese ◽  
...  
Keyword(s):  
Cancer Metabolism ◽  
Glucose Transporter ◽  
Aerobic Glycolysis ◽  
Cell Metabolism ◽  
Lactate Production ◽  
Pentose Phosphate ◽  
Metabolic Reprogramming ◽  
Effective Prevention ◽  
Anti Cancer ◽  
Glucose 6 Phosphate Dehydrogenase

Metabolic reprogramming characterized by alterations in nutrient uptake and critical molecular pathways associated with cancer cell metabolism represents a fundamental process of malignant transformation. Melatonin (N-acetyl-5-methoxytryptamine) is a hormone secreted by the pineal gland. Melatonin primarily regulates circadian rhythms but also exerts anti-inflammatory, anti-depressant, antioxidant and anti-tumor activities. Concerning cancer metabolism, melatonin displays significant anticancer effects via the regulation of key components of aerobic glycolysis, gluconeogenesis, the pentose phosphate pathway (PPP) and lipid metabolism. Melatonin treatment affects glucose transporter (GLUT) expression, glucose-6-phosphate dehydrogenase (G6PDH) activity, lactate production and other metabolic contributors. Moreover, melatonin modulates critical players in cancer development, such as HIF-1 and p53. Taken together, melatonin has notable anti-cancer effects at malignancy initiation, progression and metastasing. Further investigations of melatonin impacts relevant for cancer metabolism are expected to create innovative approaches supportive for the effective prevention and targeted therapy of cancers.

scholarly journals TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) Is Upregulated in Lymphocytes Stimulated with Concanavalin A

2021 ◽  
Vol 22 (14) ◽  
pp. 7436
Author(s):  
Helga Simon-Molas ◽  
Xavier Vallvé-Martínez ◽  
Irene Caldera-Quevedo ◽  
Pere Fontova ◽  
Claudia Arnedo-Pac ◽  
...  
Keyword(s):  
Concanavalin A ◽  
Carbon Flux ◽  
Human Lymphocytes ◽  
Tumor Development ◽  
Pentose Phosphate ◽  
Akt Signaling Pathway ◽  
G6pdh Activity ◽  
Physiological Conditions ◽  
Glucose 6 Phosphate Dehydrogenase

The glycolytic modulator TP53-Inducible Glycolysis and Apoptosis Regulator (TIGAR) is overexpressed in several types of cancer and has a role in metabolic rewiring during tumor development. However, little is known about the role of this enzyme in proliferative tissues under physiological conditions. In the current work, we analysed the role of TIGAR in primary human lymphocytes stimulated with the mitotic agent Concanavalin A (ConA). We found that TIGAR expression was induced in stimulated lymphocytes through the PI3K/AKT pathway, since Akti-1/2 and LY294002 inhibitors prevented the upregulation of TIGAR in response to ConA. In addition, suppression of TIGAR expression by siRNA decreased the levels of the proliferative marker PCNA and increased cellular ROS levels. In this model, TIGAR was found to support the activity of glucose 6-phosphate dehydrogenase (G6PDH), the first enzyme of the pentose phosphate pathway (PPP), since the inhibition of TIGAR reduced G6PDH activity and increased autophagy. In conclusion, we demonstrate here that TIGAR is upregulated in stimulated human lymphocytes through the PI3K/AKT signaling pathway, which contributes to the redirection of the carbon flux to the PPP.

Mass isotopomer study of the nonoxidative pathways of the pentose cycle with [1,2-13C2]glucose

1998 ◽  
Vol 274 (5) ◽  
pp. E843-E851 ◽  
Author(s):  
Wai-Nang Paul Lee ◽  
Laszlo G. Boros ◽  
Joaquim Puigjaner ◽  
Sara Bassilian ◽  
Shu Lim ◽  
...  
Keyword(s):  
Pentose Phosphate ◽  
Gas Chromatography Mass Spectrometry ◽  
Hep G2 ◽  
Tracer Method ◽  
Human Hepatoma Cells ◽  
Glucose Flux ◽  
Isotopomer Analysis ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Mass Isotopomer

We present a single-tracer method for the study of the pentose phosphate pathway (PPP) using [1,2-13C2]glucose and mass isotopomer analysis. The metabolism of [1,2-13C2]glucose by the glucose-6-phosphate dehydrogenase, transketolase (TK), and transaldolase (TA) reactions results in unique pentose and lactate isotopomers with either one or two13C substitutions. The distribution of these isotopomers was used to estimate parameters of the PPP using the model of Katz and Rognstad (J. Katz and R. Rognstad. Biochemistry 6: 2227–2247, 1967). Mass and position isotopomers of ribose, and lactate and palmitate (products from triose phosphate) from human hepatoma cells (Hep G2) incubated with 30% enriched [1,2-13C2]glucose were determined using gas chromatography-mass spectrometry. After 24–72 h incubation, 1.9% of lactate molecules in the medium contained one 13C substitution ( m 1) and 10% contained two 13C substitutions ( m 2). A similar m 1-to- m 2ratio was found in palmitate as expected. Pentose cycle (PC) activity determined from incubation with [1,2-13C2]glucose was 5.73 ± 0.52% of the glucose flux, which was identical to the value of PC (5.55 ± 0.73%) determined by separate incubations with [1-13C] and [6-13C]glucose.13C was found to be distributed in four ribose isotopomers ([1-13C]-, [5-13C]-, [1,2-13C2]-, and [4,5-13C2]ribose). The observed ribose isotopomer distribution was best matched with that provided from simulation by substituting 0.032 for TK and 0.85 for TA activity relative to glucose uptake into the model of Katz and Rognstad. The use of [1,2-13C2]glucose not only permits the determination of PC but also allows estimation of relative rates through the TK and TA reactions.

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