scholarly journals Investigation of Heterologously Expressed Glucose-6-Phosphate Dehydrogenase Genes in a Yeast zwf1 Deletion

2020 ◽  
Vol 8 (4) ◽  
pp. 546 ◽  
Author(s):  
Jürgen J. Heinisch ◽  
Johannes Knuesting ◽  
Renate Scheibe
Keyword(s):  
Kluyveromyces Lactis ◽  
Expression System ◽  
Disulfide Bridge ◽  
Pentose Phosphate ◽  
Yeast Saccharomyces Cerevisiae ◽  
Physiological Properties ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Synthetic Media ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme of the oxidative part of the pentose phosphate pathway and serves as the major source of NADPH for metabolic reactions and oxidative stress response in pro- and eukaryotic cells. We here report on a strain of the model yeast Saccharomyces cerevisiae which lacks the G6PD-encoding ZWF1 gene and displays distinct growth retardation on rich and synthetic media, as well as a strongly reduced chronological lifespan. This strain was used as a recipient to introduce plasmid-encoded heterologous G6PD genes, synthesized in the yeast codon usage and expressed under the control of the native PFK2 promotor. Complementation of the hypersensitivity of the zwf1 mutant towards hydrogen peroxide to different degrees was observed for the genes from humans (HsG6PD1), the milk yeast Kluyveromyces lactis (KlZWF1), the bacteria Escherichia coli (EcZWF1) and Leuconostoc mesenteroides (LmZWF1), as well as the genes encoding three different plant G6PD isoforms from Arabidopsis thaliana (AtG6PD1, AtG6PD5, AtG6PD6). The plastidic AtG6PD1 isoform retained its redox-sensitive activity when produced in the yeast as a cytosolic enzyme, demonstrating the suitability of this host for determination of its physiological properties. Mutations precluding the formation of a disulfide bridge in AtG6PD1 abolished its redox-sensitivity but improved its capacity to complement the yeast zwf1 deletion. Given the importance of G6PD in human diseases and plant growth, this heterologous expression system offers a broad range of applications.

scholarly journals The Pentose Phosphate Pathway in Yeasts–More Than a Poor Cousin of Glycolysis

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 725
Author(s):  
Laura-Katharina Bertels ◽  
Lucía Fernández Murillo ◽  
Jürgen J. Heinisch
Keyword(s):  
Pentose Phosphate Pathway ◽  
Kluyveromyces Lactis ◽  
Aromatic Amino Acids ◽  
Pentose Phosphate ◽  
Minor Role ◽  
Disease Genes ◽  
Yeast Saccharomyces Cerevisiae ◽  
Key Enzyme ◽  
A Minor ◽  
Glucose 6 Phosphate Dehydrogenase

The pentose phosphate pathway (PPP) is a route that can work in parallel to glycolysis in glucose degradation in most living cells. It has a unidirectional oxidative part with glucose-6-phosphate dehydrogenase as a key enzyme generating NADPH, and a non-oxidative part involving the reversible transketolase and transaldolase reactions, which interchange PPP metabolites with glycolysis. While the oxidative branch is vital to cope with oxidative stress, the non-oxidative branch provides precursors for the synthesis of nucleic, fatty and aromatic amino acids. For glucose catabolism in the baker’s yeast Saccharomyces cerevisiae, where its components were first discovered and extensively studied, the PPP plays only a minor role. In contrast, PPP and glycolysis contribute almost equally to glucose degradation in other yeasts. We here summarize the data available for the PPP enzymes focusing on S. cerevisiae and Kluyveromyces lactis, and describe the phenotypes of gene deletions and the benefits of their overproduction and modification. Reference to other yeasts and to the importance of the PPP in their biotechnological and medical applications is briefly being included. We propose future studies on the PPP in K. lactis to be of special interest for basic science and as a host for the expression of human disease genes.

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 Deletion of the Glucose-6-Phosphate Dehydrogenase Gene KlZWF1 Affects both Fermentative and Respiratory Metabolism in Kluyveromyces lactis

2006 ◽  
Vol 6 (1) ◽  
pp. 19-27 ◽  
Author(s):  
Michele Saliola ◽  
Gina Scappucci ◽  
Ilaria De Maria ◽  
Tiziana Lodi ◽  
Patrizia Mancini ◽  
...  
Keyword(s):  
Biomass Yield ◽  
Kluyveromyces Lactis ◽  
Carbon Sources ◽  
Pentose Phosphate ◽  
Respiratory Metabolism ◽  
Wild Type ◽  
Dehydrogenase Gene ◽  
Increased Sensitivity ◽  
Dimeric Form ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACT In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1Δ strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield.

scholarly journals Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis

2022 ◽  
Vol 23 (2) ◽  
pp. 772
Author(s):  
Rosaura Rodicio ◽  
Hans-Peter Schmitz ◽  
Jürgen J. Heinisch
Keyword(s):  
Kluyveromyces Lactis ◽  
Regulation Of Gene Expression ◽  
Carbon Sources ◽  
Pentose Phosphate ◽  
Gene Encoding ◽  
Physiological Characterization ◽  
Genes Encoding ◽  
Fructose 1,6 Bisphosphate

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

343 DISTRIBUTION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE AND IN VITRO MATURATION OF PORCINE OOCYTE - CUMULUS COMPLEXES FROM SMALL AND MEDIUM FOLLICLES

2007 ◽  
Vol 19 (1) ◽  
pp. 287
Author(s):  
Y. Ishida ◽  
H. Funahashi
Keyword(s):  
In Vitro Maturation ◽  
Critical Role ◽  
Morphological Characteristics ◽  
Cumulus Cells ◽  
Pentose Phosphate ◽  
G6pd Activity ◽  
Cresyl Blue ◽  
Key Enzyme ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose metabolism through the pentose phosphate pathway (PPP) plays a critical role in meiotic maturation and fertilization. However, the relationship between the distribution of a PPP key enzyme, glucose-6-phosphate dehydrogenase (G6PD), in cumulus–oocyte complexes (COCs) and the in vitro maturation (IVM) of the oocytes is not clear. In the present study, we examined the distribution of G6PD, the morphological characteristics in OCCs derived from small (d2 mm in diameter) and medium (3 to 6 mm in diameter) follicles, and the rate of oocyte maturation. Porcine COCs were collected from small or medium follicles of slaughterhouse ovaries. The COCs were cultured in a maturation medium, BSA-free NCSU37 supplemented with 10% porcine follicular fluid, eCG, and hCG, for 20 h and then in the absence of hormones for 24 h. To determine the distribution of G6PD, the COCs were treated with 13 �M brilliant cresyl blue (BCB) in TL-HEPES-PVA for 90 min. Results from 3–6 replicates were analyzed by ANOVA and Duncan&apos;s multiple range test. The mean diameters for COCs collected from small follicles (136.7 &micro;m for the outer zona and 103.1 &micro;m for ooplasm) were significantly less than for those derived from medium follicles (164.1 &micro;m and 122.0 &micro;m, respectively). G6PD activity was detected in the cumulus cells for most of the COCs derived from medium follicles, but it was not significantly different from that of COCs derived from small follicles. In the second group of COCs, very little G6PD activity was found in both the cumulus cells and the oocytes (34.7 &plusmn; 11.5&percnt; and 18.0 &plusmn; 6.7&percnt; in COCS derived from small and medium follicles, respectively). After stimulation by eCG and hCG, the percentages of COCS in which G6PD activity was detected in the cumulus cells, but not in the oocytes, were 56.2 &plusmn; 23.8&percnt; and 72.9 &plusmn; 6.1&percnt; for small and medium follicles, respectively. The percentage of oocytes at the metaphase II stage (53&percnt; and 63.9&percnt; in oocytes from small and medium follicles, respectively) was higher for the COCs that showed higher G6PD activity in their cumulus cells. In conclusion, although no statistical differences were detected in the distribution of G6PD between COCs from small and medium follicles, due to a large variation, a higher percentage of mature oocytes seems to be the result of COCs where the G6PD activity is detected in the cumulus cells, but not in the oocyte, during IVM.

scholarly journals Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2780-2793 ◽  
Author(s):  
K. Olavarria ◽  
J. De Ingeniis ◽  
D. C. Zielinski ◽  
M. Fuentealba ◽  
R. Muñoz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Response ◽  
Substantial Effect ◽  
Pentose Phosphate ◽  
Site Directed Mutagenesis ◽  
Dna Encoding ◽  
Genes Encoding ◽  
Balance Analysis ◽  
Glucose 6 Phosphate Dehydrogenase

In Escherichia coli, the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the major sources of NADPH when glucose is the sole carbon nutrient. However, unbalanced NADPH production causes growth impairment as observed in a strain lacking phosphoglucoisomerase (Δpgi). In this work, we studied the metabolic response of this bacterium to the replacement of its glucose-6-phosphate dehydrogenase (G6PDH) by an NADH-producing variant. The homologous enzyme from Leuconostoc mesenteroides was studied by molecular dynamics and site-directed mutagenesis to obtain the NAD-preferring LmG6PDHR46E,Q47E. Through homologous recombination, the zwf loci (encoding G6PDH) in the chromosomes of WT and Δpgi E. coli strains were replaced by DNA encoding LmG6PDHR46E,Q47E. Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect on the growth rates, increasing 59 % in the Δpgi strain, while falling 44 % in the WT. Quantitative PCR (qPCR) analysis of the zwf locus showed that the expression level of the mutant enzyme was similar to the native enzyme and the expression of genes encoding key enzymes of the central pathways also showed moderate changes among the studied strains. The phenotypic and qPCR data were integrated into in silico modelling, showing an operative G6PDH flux contributing to the NADH pool. Our results indicated that, in vivo, the generation of NADH by G6PDH is beneficial or disadvantageous for growth depending on the operation of the upper Embden–Meyerhof pathway. Interestingly, a genomic database search suggested that in bacteria lacking phosphofructokinase, the G6PDHs tend to have similar preferences for NAD and NADP. The importance of the generation of NADPH in a pathway such as the oxPPP is discussed.

Cytoplasmic glucose-6-phosphate dehydrogenase plays an important role in the silicon-enhanced alkaline tolerance in highland barley

2021 ◽  
Vol 48 (2) ◽  
pp. 119 ◽  
Author(s):  
Qiang He ◽  
Ping Li ◽  
Wenya Zhang ◽  
Yurong Bi
Keyword(s):  
Reduced Glutathione ◽  
Pentose Phosphate ◽  
Alkaline Stress ◽  
Fresh Weight ◽  
G6pdh Activity ◽  
Biotic And Abiotic Stresses ◽  
Alkaline Tolerance ◽  
Key Enzyme ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Highland Barley

Glucose-6-phosphate dehydrogenase (G6PDH), as a key enzyme in the pentose phosphate pathway, extensively responds to the biotic and abiotic stresses. In this study we focussed on the G6PDH role in the alleviation of alkaline stress induced by silicon (Si) in highland barley. Application of Si reduced the water loss and malondialdehyde (MDA) and reactive oxygen species (ROS) contents, improved the fresh weight, photosynthesis, K+ content, and the superoxide dismutase (SOD) and catalase (CAT) activities, thus alleviating the damage caused by alkaline stress. The G6PDH activity, especially the cytoplasmic G6PDH, significantly increased under alkaline stress, and was further stimulated by the addition of exogenous Si. Meanwhile, the levels of NADPH and reduced glutathione (GSH) showed similar profiles to G6PDH activity under NaHCO3 and NaHCO3 + Si treatments. The inhibition of G6PDH activity by glucosamine abolished the relieving effect of Si on alkaline stress, which was manifested in the increase of ROS and the decrease of GSH content. Together, our results suggest that Si-enhanced tolerance of alkaline stress may be related to the regulation of GSH levels by the cytoplasmic G6PDH in highland barley.

scholarly journals Isolating an active and inactive CACTA transposon from lettuce color mutants and characterizing their family

2021 ◽  
Author(s):  
Csanad Gurdon ◽  
Alexander Kozik ◽  
Rong Tao ◽  
Alexander Poulev ◽  
Isabel Armas ◽  
...  
Keyword(s):  
Anthocyanin Biosynthesis ◽  
Mobile Element ◽  
Bioinformatic Analysis ◽  
Relative Expression Level ◽  
Gene Coding ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Natural Derivative ◽  
Dark Green ◽  
Transposon Insertions

Abstract Dietary flavonoids play an important role in human nutrition and health. Flavonoid biosynthesis genes have recently been identified in lettuce (Lactuca sativa); however, few mutants have been characterized. We now report the causative mutations in Green Super Lettuce (GSL), a natural light green mutant derived from red cultivar NAR; and GSL-Dark Green (GSL-DG), an olive-green natural derivative of GSL. GSL harbors CACTA 1 (LsC1), a 3.9-kb active nonautonomous CACTA superfamily transposon inserted in the 5′ untranslated region of anthocyanidin synthase (ANS), a gene coding for a key enzyme in anthocyanin biosynthesis. Both terminal inverted repeats (TIRs) of this transposon were intact, enabling somatic excision of the mobile element, which led to the restoration of ANS expression and the accumulation of red anthocyanins in sectors on otherwise green leaves. GSL-DG harbors CACTA 2 (LsC2), a 1.1-kb truncated copy of LsC1 that lacks one of the TIRs, rendering the transposon inactive. RNA-sequencing and reverse transcription quantitative PCR of NAR, GSL, and GSL-DG indicated the relative expression level of ANS was strongly influenced by the transposon insertions. Analysis of flavonoid content indicated leaf cyanidin levels correlated positively with ANS expression. Bioinformatic analysis of the cv Salinas lettuce reference genome led to the discovery and characterization of an LsC1 transposon family with a putative transposon copy number greater than 1,700. Homologs of tnpA and tnpD, the genes encoding two proteins necessary for activation of transposition of CACTA elements, were also identified in the lettuce genome.

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.

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