Human Glucose 6-phosphate Dehydrogenase Research Articles

Novel Humanized G6PD Deficient Mice Reveal Mechanisms of Increased Tolerance to Exercise As Gleaned By Multi-Omics in Models of Critical Speed

INTRODUCTION: Glucose-6-phosphate-dehydrogenase (G6PD) deficiency is the most common enzymatic deficiency in humans, affecting ~500 million people or ~6% of mankind. G6PD is the rate limiting enzyme of the pentose phosphate pathway (PPP), the main antioxidant pathway in mature red blood cells (RBCs). Activation of the PPP generates NADPH, an essential reducing equivalent for antioxidant metabolism that protects against intra- and extra-vascular hemolysis in response to oxidant stress. Impaired G6PD activity makes RBCs susceptible to hemolysis induced by oxidant challenges. Exercise-induced oxidant stress is a well-established concept in the field of physiology. Individuals with G6PD deficiency are recommended to refrain from strenuous exercise, as they are deemed to be at increased risk for hemolytic events in response to exercise-induced oxidant stress. While biochemically sound, this rationale has never been tested mechanistically, owing to ethical and technical limitations. As such, knowledge is limited regarding molecular impacts of exercise activity in G6PD deficient individuals. METHODS: Here we generated novel humanized mouse models carrying either (i) human canonical G6PD (normal activity), or two of the most common G6PD variants (ii) African (V68M - < 10% residual enzymatic activity) or (iii) Mediterranean (S188F - <1% residual enzymatic activity). We leveraged these mouse models to assess critical speed (CS), a functional measurement of the speed-to-duration relationship. To expand the molecular resolution of genetically associated adaptations to exercise, CS tests were combined with measurements of hemodynamics - including cardiac output, imaging (e.g., scanning electron microscopy of RBCs before and after exercise) and multi-omics (mass spectrometry-based metabolomics, lipidomics and proteomics) characterization of plasma, RBCs, and multiple tissue matrices including liver, heart, kidney, lung, spleen, and muscles (gastrocnemius and soleus). RESULTS: First of all, we confirmed a significant decrease in G6PD activity in RBCs from mice carrying the African and Mediterranean variants ( Figure 1). Unexpectedly, G6PD deficient mice showed ~10% faster CS compared to mice carrying the canonical human G6PD (p<0.0001 and p<0.02, Figure 1). While scanning electron microscopy showed differences before and after exercise, with accumulation of reversibly modified morphologies across all groups after the run, no significant differences were observed across groups. No significant changes in hemolysis markers were observed in plasma samples after exercise, neither at the metabolic (e.g., heme catabolites) or proteomics level (e.g., hemoglobin chains, RBC-derived proteins). Within hemodynamics, the hG6PD Med- mice are showing a higher efficiency in their cardiac function through cardiac function (p<0.005 - Figure 1). Pathway analysis of metabolomics data generated from RBC and plasma fractions showed significant decreases in arginine metabolism, increased glycolysis, and elevated Krebs cycle intermediates, in addition to expected differences in PPP. Specifically, in hG6PD A- RBC acylcarnitines accumulate after running. In hG6PD Med-, there are clear left ventricular differences highlighted in energy metabolism ( Figure 1). Multiomics analyses identified five major tissue clusters between heart, muscle, liver, kidney, and spleen, with G6PD function having the most significant metabolic impact on liver and soleus. RBC proteomics results highlighted deceased ferroportin (key iron transporter) in both the deficient groups, with markedly significant changes in the hG6PD Med- (p<0.0001). CONCLUSION: Novel humanized G6PD deficient mice maintain faster CS compared to mice. Omics approaches afford the opportunity to identify metabolic correlates to measurements of performance in relevant animal models, such as the CS model, which offer expanded mechanistic understanding of molecular bases of CS and the associated impact of G6PD deficiency. Our results not only challenge the assumption that exercise-induced oxidant stress predisposes the ~500M individuals with G6PD deficiency worldwide to increased hemolytic propensity, but rather indicate a metabolic advantage of functional polymorphisms to G6PD with respect to exercise performance, an observation that could contribute explaining the prevalence of such mutation in human populations.

Virtual prediction of fava bean (Vicia faba L.) active compounds to inhibit G6PD enzyme activity

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common hereditary enzyme defect in humans, caused by a mutation in the X-linked gene encoding G6PD. The G6PD enzyme plays an important role to produced reducing agents which maintain reduced glutathione through pentose phosphate pathway. On ingestion of Faba Bean (Vicia faba L.), vicine and convicine are hydrolyzed by β -glucosidase to divicine and isouramil which can cause acute hemolytic anemia in patients with G6PD deficiency. This study aims to determine the molecular mechanism of bioactive compounds from fava beans as an inhibitor of the human G6PD Canton enzyme activity through an in silico approach. Screening for toxic bioactive compounds of V. faba was carried out using Swiss ADME. Molecular docking was performed to identify the interaction of divicine and isouramil binding with human G6PD Canton. Dobutamine hydrochloride was used as a negative control ligand. The result of Swiss ADME screening showed that convicine and vicine were not qualified for the Lipinski rules of five. Docking analysis demonstrated similar binding interaction of divicine and isouramil compared with control ligand to bound G6PD canton allosteric sites and shift the binding position of NADP+, suggesting these compounds interrupt the NADP+ to produce NADPH. The binding affinity of divicine (-7.0 kcal/mol) was lower than dobutamine hydrochloride (-6.9 kcal/mol), while isouramil was higher (-6.5 kcal/mol). Accordingly, this study was pointed out that divicine and isouramil have the potential as inhibitor of G6PD enzyme activity. Further, in vivo and in vitro studies are needed to confirm this research.

Allosteric role of a structural NADP+ molecule in glucose-6-phosphate dehydrogenase activity

Human glucose-6-phosphate dehydrogenase (G6PD) is the main cellular source of NADPH, and thus plays a key role in maintaining reduced glutathione to protect cells from oxidative stress disorders such as hemolytic anemia. G6PD is a multimeric enzyme that uses the cofactors β-D-glucose 6-phosphate (G6P) and "catalytic" NADP+ (NADP+c), as well as a "structural" NADP+ (NADP+s) located ∼25 Å from the active site, to generate NADPH. While X-ray crystallographic and biochemical studies have revealed a role for NADP+s in maintaining the catalytic activity by stabilizing the multimeric G6PD conformation, other potential roles for NADP+s have not been evaluated. Here, we determined the high resolution cryo-electron microscopy structures of human wild-type G6PD in the absence of bound ligands and a catalytic G6PD-D200N mutant bound to NADP+c and NADP+s in the absence or presence of G6P. A comparison of these structures, together with previously reported structures, reveals that the unliganded human G6PD forms a mixture of dimers and tetramers with similar overall folds, and binding of NADP+s induces a structural ordering of a C-terminal extension region and allosterically regulates G6P binding and catalysis. These studies have implications for understanding G6PD deficiencies and for therapy of G6PD-mediated disorders.

The splicing factor 9G8 regulates the expression of NADPH-producing enzyme genes in Drosophila

Excess nutrients are stored as triglycerides, mostly as lipid droplets found in adipose tissue. Previous studies have characterized a group of splicing factors called serine/arginine rich (SR) proteins that function to identify intron/exon borders in regulating metabolic homeostasis in the Drosophila fat body. Decreasing the function of one SR protein, 9G8, causes an increase in triglyceride storage; however, the full complement of genes regulated by 9G8 to control metabolism is unknown. To address this question, we performed RNA sequencing on Drosophila fat bodies with 9G8 levels reduced by RNAi. Differential expression and differential exon usage analyses revealed several genes involved in the immune response, xenobiotic biology, protein translation, sleep, and lipid and carbohydrate metabolism whose expression or splicing is altered in 9G8-RNAi fat bodies. One gene that was both downregulated and had altered splicing in 9G8-RNAi fat bodies was Zwischenferment (Zw), the Drosophila homolog of human glucose 6-phosphate dehydrogenase (G6PD). G6PD regulates flux of glucose 6-phosphate (G6P) into the pentose phosphate pathway, which generates NADPH, a coenzyme for lipid synthesis. Interestingly, the other NADPH-producing enzyme genes in Drosophila (phosphogluconate dehydrogenase, isocitrate dehydrogenase and malic enzyme) were also decreased in 9G8-RNAi flies. Together, these findings suggest that 9G8 regulates several classes of genes and may regulate NADPH-producing enzyme genes to maintain metabolic homeostasis.

An Optimized Dihydrodibenzothiazepine Lead Compound (SBI-0797750) as a Potent and Selective Inhibitor of Plasmodium falciparum and P. vivax Glucose 6-Phosphate Dehydrogenase 6-Phosphogluconolactonase

In Plasmodium, the first two and rate-limiting enzymes of the pentose phosphate pathway, glucose 6-phosphate dehydrogenase (G6PD) and the 6-phosphogluconolactonase, are bifunctionally fused to a unique enzyme named GluPho, differing structurally and mechanistically from the respective human orthologs. Consistent with the enzyme's essentiality for malaria parasite proliferation and propagation, human G6PD deficiency has immense impact on protection against severe malaria, making PfGluPho an attractive antimalarial drug target. Herein we report on the optimized lead compound N-(((2R,4S)-1-cyclobutyl-4-hydroxypyrrolidin-2-yl)methyl)-6-fluoro-4-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide (SBI-0797750), a potent and fully selective PfGluPho inhibitor with robust nanomolar activity against recombinant PfGluPho, PvG6PD, and P. falciparum blood-stage parasites. Mode-of-action studies have confirmed that SBI-0797750 disturbs the cytosolic glutathione-dependent redox potential, as well as the cytosolic and mitochondrial H2O2 homeostasis of P. falciparum blood stages, at low nanomolar concentrations. Moreover, SBI-0797750 does not harm red blood cell (RBC) integrity and phagocytosis and thus does not promote anemia. SBI-0797750 is therefore a very promising antimalarial lead compound.

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

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.

Hematologic and systemic metabolic alterations due to Mediterranean class II G6PD deficiency in mice.

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (approximately 5%). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of WT stable G6PD caused by promoter mutations. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells (RBCs) that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency, Med(-), was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs, and deficient mice had increased hemolytic sequelae to oxidant stress. Nonerythroid organs had metabolic changes consistent with mild G6PD deficiency, consistent with what has been observed in humans. Juxtaposition of G6PD-deficient and WT mice revealed altered lipid metabolism in multiple organ systems. Together, these findings both establish a mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and advance our basic understanding of altered metabolism in this setting.

Kinetic Efficiency of R252L and D60N Mutants of C. elegans Glucose‐6‐Phosphate Dehydrogenase Relative to the Wild Type

Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme present in all species that have thus far been tested (Vuillamy et al., 1988). G6PD is the rate limiting enzyme in the pentose phosphate pathway (PPP) (Wang and Engel, 2009) and reduces NADP+ to NADPH through the oxidation of glucose-6-phosphate (G6P) to 6-phospho-gluconolactone (Hirono et al. 1989). The effectiveness of the G6PD enzyme is vital to the health and survival of an organism, as the PPP is one of the major NADPH production mechanisms, and NADPH protects cells from succumbing to oxidative stress (Wang and Engel, 2009). In the present study, kinetic characterization of the C. elegans orthologs of two known deficient human G6PD variants was performed. Production of the R252L and D60N C. elegans G6PD variants was done via E. coli plasmid expression. Purification of the mutant enzymes was performed on a nickel column, and enzyme kinetics parameters of the mutant G6PD enzymes were determined and compared to those for the wild type enzyme. From the Michaelis-Menten kinetics assay results, it was determined that the Vmax value for the R252L mutant enzyme was 5.9 * 10-3 ± 7.5 * 10-4 µmol/min, the KM value was 1050 ± 200 µM, and the specific activity was 1.2 µmol/min/mg. For the D60N enzyme variant it was determined that the Vmax value was 3.2 * 10-3 ± 2.1 * 10 -4 µmol/min, the KM value was 80 ± 22 µM, and the specific activity was 6.5 µmol/min/mg. Lastly, the Vmax value for the wild type enzyme was 2.2 * 10-3 ± 1.4 * 10 -4 µmol/min, the KM value was 100 ± 27 µM, and the specific activity was 11.1 µmol/min/mg. These results diverge slightly from those previously published on the human orthologs of the two mutants. A 2013 study by Bendaoud et al. in which the human ortholog of the R252L GSPD mutant was first cited, as G6PD Tunisia, found that the mutant retained an estimated 25% of normal enzymatic activity while this study found that the R252L mutant had about 11% activity compared to the wild type. A 1988 study by Vuillamy et al. found that the Metaponto variant (the human ortholog of the D60N mutant) had a KM of 47 µM relative to G6P concentration, which is a little over half that observed in this study. The same study found that the enzymatic activity of the mutant within red blood cells was between 14 and 39% of the wild type while the present study found the activity of the D60N mutant to be about 59% of the wild type. This novel data presents evidence of the deficiency of the C. elegans orthologs of the Tunisia and Metaponto variants of human G6PD, thus illustrating their promise as model systems for their human counterparts. Bendaoud B, Hosni I, Mosbahi I, Hafsia R, Prehu C, Abbes S. 2013. Pathol Biol (Paris). 61(2):64–69. Hirono A, Kuhl W, Gelbart T, Forman L, Fairbanks VF, Beutler E. 1989. Proc Natl Acad Sci USA. 86(24):10015–10017. Vulliamy TJ, D'Urso M, Battistuzzi G, Estrada M, Foulkes NS, Martini G, Calabro V, Poggi V, Giordano R, Town M. 1988. Proc Natl Acad Sci U S A. 85(14):5171–5175. Wang X-T, Engel PC. 2009. Biochim Biophys Acta. 1792(8):804–809.

Modeling human glucose-6-phosphate dehydrogenase mutations using C. elegans GSPD-1.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked, recessive condition that causes intermittent jaundice or hemolytic anemia because of low NADPH levels in red blood cells. We performed steady-state enzyme kinetics with the recombinant C. elegans ortholog of human G6PD, GSPD-1, and two mutants containing amino acid changes found in human patients. The KM values for glucose-6-phosphate were 100 ± 27 µM, 80 ± 22 µM, and 1000 ± 300 µM for the wild-type, D60N, and R252L GSPD-1 enzymes, respectively. The specific activities of the D60N and R252L mutants were 59% and 11%, respectively, of the wild-type value. Protein homology modeling suggested that the R252L mutation was more severe because the mutation caused a shift in the position of some active site residues. The D60N mutation may have affected the conformation of an outer loop of the enzyme. These data demonstrate that GSPD-1 is a promising model for human G6PD deficiencies, with the advantage that potential treatments could be studied in vivo in C. elegans.

Production and differential activity of recombinant human wild-type G6PD and G6PDViangchan.

Glucose-6-phosphate dehydrogenase (G6PD) is essential to produce reduced nicotinamide adenine dinucleotide phosphate, which is required to protect cells against oxidative stress. G6PD deficiency is a genetic variation that may lead to hemolysis with potential consequences, such as kidney failure, and patients often experience low quality of life. To establish a simple, efficient, and optimized method to produce a G6PDViangchan variant and characterize the phenotypes of recombinant human wild-type G6PD and G6PDViangchan. G6PD was amplified by polymerase chain reaction (PCR) from a human cDNA plasmid, and the gene for G6PDViangchan was amplified by initiating a mutation at location 871 (G>A) through site-directed mutagenesis. Protein expression and western blotting were conducted after successful cloning. The enzymatic activity of both proteins was assessed spectrophotometrically after purification. Both amplicons were successfully cloned into a pET26b(+) expression vector and transformed into Escherichia coli BL21 (DE3) cells for overexpression as C-terminally histidine-tagged recombinant proteins. Western blotting confirmed that both proteins were successfully produced at similar levels. The enzymes were purified by immobilized metal (Co) affinity chromatography. Postpurification assay of enzyme activity revealed about 2-fold differences in the levels of specific activity between the wild-type G6PD (155.88 U/mg) and G6PDViangchan (81.85 U/mg), which is consistent with earlier reports. Analysis in silico showed that the coding change in G6PDViangchan has a substantial effect on protein folding structure. We successfully cloned, expressed, and purified both wild-type G6PD and G6PDViangchan proteins. Such a protocol may be useful for creating a model system to study G6PD deficiency disease.

Functional and structural analysis of double and triple mutants reveals the contribution of protein instability to clinical manifestations of G6PD variants

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common polymorphism and enzymopathy in humans, affecting approximately 400 million people worldwide. Over 200 point mutations have been identified in g6pd and the molecular mechanisms underlying the severity of G6PD variants differ. We report the detailed functional and structural characterization of 11 recombinant human G6PD variants: G6PD Asahi, G6PD A, G6PD Guadalajara, G6PD Acrokorinthos, G6PD Ananindeua, G6PD A-(202), G6PD Sierra Leone, G6PD A-(680), G6PD A-(968), G6PD Mount Sinai and G6PD No name. G6PD Guadalajara, G6PD Mount Sinai and G6PD No name are inactive variants and, correlating with the observed clinical manifestations, exhibit complete loss of enzyme activity. Protein structural instability, causing a reduction in catalytic efficiency, contributes to the clinical phenotypes of all variants. In double and triple mutants sharing the G6PD A mutation, we observed cooperative interaction between two and three mutations to cause protein dysfunction. The G6PD A (Asn126Asp) mutation exhibits no effect on protein activity and stability, indicating that the additional mutations in these G6PD variants significantly contribute to enzyme deficiency. We provide insight into the molecular basis of G6PD deficiency, which can explain the severity of clinical manifestations observed in individuals with G6PD deficiency.

Development of Selective Steroid Inhibitors for the Glucose-6-phosphate Dehydrogenase from Trypanosoma cruzi.

Chagas disease is a parasitic infection affecting millions of people across Latin America, imposing a dramatic socioeconomic burden. Despite the availability of drugs, nifurtimox and benznidazole, lack of efficacy and incidence of side-effects prompt the identification of novel, efficient, and affordable drug candidates. To address this issue, one strategy could be probing the susceptibility of Trypanosoma parasites toward NADP-dependent enzyme inhibitors. Recently, steroids of the androstane group have been described as highly potent but nonselective inhibitors of parasitic glucose-6-phosphate dehydrogenase (G6PDH). In order to promote selectivity, we have synthesized and evaluated 26 steroid derivatives of epiandrosterone in enzymatic assays, whereby 17 compounds were shown to display moderate to high selectivity for T. cruzi over the human G6PDH. In addition, three compounds were effective in killing intracellular T. cruzi forms infecting rat cardiomyocytes. Altogether, this study provides new SAR data around G6PDH and further supports this target for treating Chagas disease.

Evaluation of the Functional Effects of an African American Glucose-6-Phosphate Dehydrogenase (G6PD) Polymorphism (Val68Met) on RBC Hemolytic Propensity and Post-Transfusion Recovery in a Humanized Mouse Model

Background: Growing evidence suggest that donor genetic variation is associated with RBCs storage integrity and post-transfusion recovery. In collaboration with the REDS III program, we performed a large-scale genome-wide association study (GWAS) study in ~13,000 healthy human blood donors, which demonstrated that RBCs with an African American G6PD-deficient A- variant (rs1050828, Val68Met) was associated with higher oxidative hemolysis after cold storage in normal volunteers. Despite a high prevalence of X-linked G6PD mutation in African American population (> 10%), blood donors are not routinely checked for G6PD deficiency and its importance in transfusion medicine is relatively understudied. We hypothesize that G6PD V68M SNP is associated with G6PD deficiency and modulates RBCs in vitro hemolytic propensity and in vivo post-transfusion recovery. Methods and Results: CRISPR-Cas9 technology was used to create non-synonymous human G6PD X-linked SNP (Val68Met) in C57B6 mice, and the desired genotypes were isolated by strategic back-crossing and sequential genotyping to ensure passage of SNP. G6PD enzymatic activity in erythrocytes was measured in fresh collected blood using a colorimetric assay kit. The predisposition of fresh and stored RBCs (after 11 days cold storage) to hemolysis was evaluated by subjecting washed mouse RBCs to selected stress assays, including osmotic fragility, mechanical fragility, and oxidative hemolysis using AAPH, diamide or H2O2. Hemolysis was measured by detection of supernatant cell-free hemoglobin using Drabkin's assay. Hematological values were measured using a Hemavet® 950 Hematology Analyzer System. Reticulocyte count was obtained using thiazole orange staining and analyzed by flow cytometry. We found severe disruption of G6PD enzymatic activity in erythrocytes from G6PD V68M SNP mice compared to WT mice (~5% residual activity in hemizygous male and ~60% in heterozygous female mice). Significant increased oxidative hemolysis was observed in both fresh and stored mouse RBCs with G6PD SNP, consistent with the GWAS study in human. G6PD V68M SNP hemizygous male mice had higher mean corpuscular volume (MCV) and lower mean corpuscular hemoglobin concentration (MCHC) compared to WT mice. However, no difference was observed in storage hemolysis, osmotic fragility, mechanical fragility and reticulocyte counts. Transfusion experiments with stored red blood cells from G6PD hemizygote males into GFP positive recipients will evaluate red blood cell recovery and half-life after standard cold storage and transfusion. Conclusions: We successfully generated a novel mouse strain carrying a "humanized" African American G6PD V68M variant which resembles the phenotype of humans with G6PD deficiency and increased oxidative hemolysis. Studies are undertaken to further investigate the effects of G6PD V68M SNP on RBCs structure, functions and in vivo post-transfusion recovery. Disclosures Gladwin: Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning.

Discovery of a novel small‐molecule activator that corrects G6PD deficiency

Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic disorders, is caused by over 160 different point mutations and results in a variety of health problems, including hemolytic anemia and neurological damage. As G6PD is a major source of protective anti‐oxidant through NADPH production, G6PD deficiency likely contributes to the severity of many other acute and chronic diseases associated with increased oxidative stress. However, no medications are available to treat G6PD deficiency directly; thus, we sought to identify a small molecule that increases catalysis of G6PD mutant enzymes. Crystal structure and mutagenesis analyses of one common mutant (Canton, R459L) provided insight into the functional defect of the enzyme. Using high‐throughput screening, we identified AG1, a small molecule that increases the activity and/or stability of the wild‐type, the Canton mutant and several other common human G6PD mutants as well. AG1 reduced oxidative stress and increased NADPH levels in vivo, in zebrafish, and prevented chloroquine‐induced hemolysis in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the clinical problems associated with multiple variants of G6PD deficiency.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glucose-6-Phosphate Dehydrogenase from the Human Pathogen Trypanosoma cruzi Evolved Unique Structural Features to Support Efficient Product Formation

Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme supplying reducing power (NADPH) to the cells, by oxidation of glucose-6-phosphate (G6P), and in the process providing a precursor of ribose-5-phosphate. G6PDH is also a virulence factor of pathogenic trypanosomatid parasites. To uncover the biochemical and structural features that distinguish TcG6PDH from its human homolog, we have solved and analyzed the crystal structures of the G6PDH from Trypanosoma cruzi (TcG6PDH), alone and in complex with G6P. TcG6PDH crystallized as a tetramer and enzymatic assays further indicated that the tetramer is the active form in the parasite, in contrast to human G6PDH, which displays higher activity as a dimer. This quaternary structure was shown to be particularly stable. The molecular reasons behind this disparity were unveiled by structural analyses: a TcG6PDH-specific residue, R323, is located at the dimer–dimer interface, critically contributing with two salt bridges per subunit that are absent in the human enzyme. This explains why TcG6PDH dimerization impaired enzyme activity. The parasite protein is also distinct in displaying a 37-amino-acid extension at the N-terminus, which comprises the non-conserved C8 and C34 involved in the covalent linkage of two neighboring protomers. In addition, a cysteine triad (C53, C94 and C135) specific of Kinetoplastid G6PDHs proved critical for stabilization of TcG6PDH active site. Based on the structural and biochemical data, we posit that the N-terminal region and the catalytic site are highly dynamic. The unique structural features of TcG6PDH pave the way toward the design of efficacious and highly specific anti-trypanosomal drugs.

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

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.

Cloning and biochemical characterization of three glucose‑6‑phosphate dehydrogenase mutants presents in the Mexican population

The deficiency of glucose‑6‑phosphate dehydrogenase (G6PD) is one of the most common inborn errors of metabolism worldwide. This congenital disorder generally results from mutations that are spread throughout the entire gene of G6PD. Three single-point mutations for G6PD have been reported in the Mexican population and named Veracruz (Arg365His), G6PD Seattle (Asp282His), and G6PD Mexico DF (Thr65Ala), whose biochemical characterization have not yet been studied. For this reason, in this work we analyzed the putative role of the three mutations to uncover the functional consequences on G6PD activity. To this end, was developed a method to clone, overexpress, and purify recombinant human G6PD. The results obtained from all variants showed a loss of catalysis by 80 to 97% and had a decrease in affinity for both physiological substrates with respect to the wild type (WT) G6PD. Our results also showed that the three mutations affected three-dimensional structure and protein stability, suggesting an unstable structure with low conformational stability that affected its G6PD functionality. Finally, based on the biochemical characterization of the unclassified G6PD Mexico DF, we suggest that this variant could be grouped as a Class I variant, because biochemical data are similar with other Class I G6PDs.

Discovery of novel glucose‐6‐phosphate dehydrogenase activators to correct G6PD deficiency

Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic disorders, is caused by over 160 different point mutations and results in a variety of health problems, including hemolytic anemia and neurological damage. As G6PD is a major source of protective anti‐oxidant through NADPH production, G6PD deficiency likely contributes to the severity of many other acute and chronic diseases associated with increased oxidative stress. As no medications are available to treat G6PD deficiency directly, we sought to identify a small molecule that increases catalysis of G6PD mutant enzymes. Crystal structure and mutagenesis analyses of one common mutant (Canton, R459L) provided insight into the functional defect of the enzyme. Using high‐throughput screening, we identified AG1, a small molecule that increases the activity of the wild‐type, the Canton mutant and several other common human G6PD mutants as well. AG1 reduced oxidative stress and increased NADPH levels in vivo, in zebrafish, and prevented chloroquine‐induced hemolysis of human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the clinical problems associated with multiple variants of G6PD deficiency.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glucose-6-phosphate dehydrogenase is indispensable in embryonic development by modulation of epithelial-mesenchymal transition via the NOX/Smad3/miR-200b axis

Glucose-6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme involved in the pentose phosphate shunt for producing nicotinamide adenine dinucleotide phosphate (NADPH). Severe G6PD deficiency leads to embryonic lethality, but the underlying mechanism is unclear. In the current study, the effects of G6PD on epithelial–mesenchymal transition (EMT), especially during embryonic development, were investigated. The knockdown of G6PD induced morphological changes, accompanied by the suppression of epithelial markers, E-cadherin and β-catenin, in A549 and MDCK cells. Such modulation of EMT was corroborated by the enhancement of migration ability in G6PD-knockdown A549 cells. Zebrafish embryos with g6pd knockdown exhibited downregulation of the E-cadherin/β-catenin adhesion molecules and impaired embryonic development through reduction in epiboly rate and increase in cell shedding at the embryo surface. The dysregulation in zebrafish embryonic development caused by g6pd knockdown could be rescued through human G6PD or CDH1 (E-cadherin gene) cRNA coinjection. The Smad3/miR-200b axis was dysregulated upon G6PD knockdown, and the reconstitution of SMAD3 in G6PD-knockdown A549 cells restored the expression of E-cadherin/β-catenin. The inhibition of NADPH oxidase (NOX) activation through the loss of p22phox signaling was involved in the dysregulation of the Smad3/miR-200b axis upon G6PD knockdown. The reconstitution of G6PD led to the recovery of the regulation of NOX/Smad3/miR-200b signaling and increased the expression of E-cadherin/β-catenin in G6PD-knockdown cells. Thus, these results suggest that in the EMT process, G6PD plays an important regulatory role as an integral component of the NOX/Smad3/miR-200b axis.

Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa

Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.363) plays an important role in the human pathogen Pseudomonas aeruginosa because it generates NADPH, an essential cofactor for several biosynthetic pathways and antioxidant enzymes. P. aeruginosa G6PDH is also a key enzyme in the metabolism of various carbon sources, such as glucose, glycerol, fructose, and mannitol. Understanding the kinetic characteristics and mechanisms that control the activity of this enzyme is crucial for future studies in this context. However, one of the impediments to achieving this goal is the limited amount of protein obtained when current purification protocols are implemented, a factor curtailing its biochemical characterization. In this study, we report a fast, efficient and reproducible procedure for the purification of P. aeruginosa G6PDH that can be implemented in a short period (2 days). In order to establish this protocol, the zwf gene, which encodes for this enzyme, was cloned and overexpressed in Escherichia coli cells. In contrast to other procedures, our method is based on protein precipitation with CaCl2 and further purification by ion exchange chromatography. Using this protocol, we were able to obtain 31 mg/L of pure protein that manifested specific activity of 145.7 U/mg. The recombinant enzyme obtained in this study manifested similar physicochemical and kinetic properties to those reported in previous works for this molecule. The large quantities of active enzyme obtained using this procedure will facilitate its structural characterization and identify differences between P. aeruginosa- and human G6PDH, thus contributing to the search for selective inhibitors against the bacterial enzyme.