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 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.

Antibacterial Mechanisms of Orostachys Cartilaginous Cell Cultures: Effect on Cell Permeability and Respiratory Metabolism of Bacillis Subtilis

2021 ◽  
Author(s):  
Y. X. Liu ◽  
X. L. Jiang ◽  
Y. N. Xu ◽  
X. C. Piao ◽  
Mei-Lan Lian
Keyword(s):  
Cell Cultures ◽  
Cell Damage ◽  
Underlying Mechanism ◽  
Iodoacetic Acid ◽  
Pentose Phosphate ◽  
Cell Permeability ◽  
Respiratory Metabolism ◽  
Mountain Area ◽  
Bacillis Subtilis ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract Orostachys cartilaginous of Crassulaceae family is a plant native to the Changbai Mountain area, China. Although O. cartilaginous has various medicinal values, its product development and production are restricted by the insufficient resource available. O. cartilaginous cell cultures possess an efficient antibacterial effect against Bacillis subtilis, but the underlying mechanism is not clear yet. Therefore, this study investigated the effects of extract from bioreactor cultured O. cartilaginous cells (OE) on B. subtilis cell permeability and respiratory metabolism to provide a reference for the further utilization of O. cartilaginous cell cultures. Results showed alkaline phosphatase activity, electrical conductivity, nucleic acid and protein contents in B. subtilis suspensions were significantly increased (p < 0.01) by OE treatment, indicating the occurrence of cell damage or increase in cell permeability. OE inhibited B. subtilis respiration, and the combination groups of OE+iodoacetic acid (IA) and OE+sodium phosphate (SP) showed low superposition rates (approximately 35%), revealing that OE likely affected IA- and SP-represented metabolic pathways. The activities of B. subtilis enzymes, specifically, hexokinase and pyruvate kinase in the Embden-Meyerhof (EMP) pathway and glucose-6-phosphate dehydrogenase in the pentose phosphate (HMP) pathway, decreased after OE treatment. This result proved that OE inhibited B. subtilis respiration by regulating the EMP and HMP pathways.

scholarly journals NHERF1 Loss Upregulates Enzymes of the Pentose Phosphate Pathway in Kidney Cortex

Antioxidants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 862
Author(s):  
Adrienne Bushau-Sprinkle ◽  
Michelle T. Barati ◽  
Kenneth B. Gagnon ◽  
Syed Jalal Khundmiri ◽  
Kathleen Kitterman ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Mitochondrial Protein ◽  
Pentose Phosphate ◽  
Metabolic Enzyme ◽  
Kidney Cortex ◽  
Mitochondrial Morphology ◽  
Proximal Tubule Cells ◽  
Cisplatin Nephrotoxicity ◽  
Increased Sensitivity ◽  
Glucose 6 Phosphate Dehydrogenase

(1) Background: We previously showed Na/H exchange regulatory factor 1 (NHERF1) loss resulted in increased susceptibility to cisplatin nephrotoxicity. NHERF1-deficient cultured proximal tubule cells and proximal tubules from NHERF1 knockout (KO) mice exhibit altered mitochondrial protein expression and poor survival. We hypothesized that NHERF1 loss results in changes in metabolic pathways and/or mitochondrial dysfunction, leading to increased sensitivity to cisplatin nephrotoxicity. (2) Methods: Two to 4-month-old male wildtype (WT) and KO mice were treated with vehicle or cisplatin (20 mg/kg dose IP). After 72 h, kidney cortex homogenates were utilized for metabolic enzyme activities. Non-treated kidneys were used to isolate mitochondria for mitochondrial respiration via the Seahorse XF24 analyzer. Non-treated kidneys were also used for LC-MS analysis to evaluate kidney ATP abundance, and electron microscopy (EM) was utilized to evaluate mitochondrial morphology and number. (3) Results: KO mouse kidneys exhibit significant increases in malic enzyme and glucose-6 phosphate dehydrogenase activity under baseline conditions but in no other gluconeogenic or glycolytic enzymes. NHERF1 loss does not decrease kidney ATP content. Mitochondrial morphology, number, and area appeared normal. Isolated mitochondria function was similar between WT and KO. Conclusions: KO kidneys experience a shift in metabolism to the pentose phosphate pathway, which may sensitize them to the oxidative stress imposed by cisplatin.

scholarly journals Importance of glucose-6-phosphate dehydrogenase in the adaptive response to hydrogen peroxide in Saccharomyces cerevisiae

1998 ◽  
Vol 330 (2) ◽  
pp. 811-817 ◽  
Author(s):  
Shingo IZAWA ◽  
Keiko MAEDA ◽  
Takeo MIKI ◽  
Junichi MANO ◽  
Yoshiharu INOUE ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
Adaptive Response ◽  
Yeast Cells ◽  
Wild Type ◽  
Total Glutathione ◽  
Dependent Activity ◽  
Increased Sensitivity ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Increased Susceptibility

Glucose-6-phosphate dehydrogenase (G6PDH)-deficient cells of Saccharomyces cerevisiae showed increased susceptibility and were unable to induce adaptation to oxidative stress. Historically, mainly in human erythrocytes, it has been suggested and accepted that decreased cellular GSH, due to loss of the NADPH-dependent activity of glutathione reductase (GR), is responsible for the increased sensitivity to oxidative stress in G6PDH-deficient cells. In the present study we investigated whether the increased susceptibility and the inability to induce adaptation to H2O2 stress of G6PDH-deficient yeast is caused by incompleteness of glutathione recycling. We constructed G6PDH- and GR-deficient mutants and analysed their adaptive response to H2O2 stress. Although G6PDH-deficient cells contained comparable amounts of GSH and GR activity to wild-type cells, GSSG was not reduced efficiently, and intracellular GSSG levels and the ratio of GSSG to total glutathione (GSSG/tGSH) were higher in G6PDH-deficient cells than in wild-type. On the other hand, GR-deficient cells showed a susceptibility identical with that of wild-type cells and induced adaptation to H2O2 stress, even though the GSSG/tGSH ratio in GR-deficient cells was higher than in G6PDH-deficient cells. These results indicate that incompleteness of glutathione recycling alone is not sufficient to account for the increased sensitivity and inability to induce adaptation to H2O2 stress of G6PDH-deficient yeast cells. In S. cerevisiae, G6PDH appears to play other important roles in the adaptive response to H2O2 stress besides supplying NADPH to the GR reaction.

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.

scholarly journals Engineering of Primary Carbon Metabolism for Improved Antibiotic Production in Streptomyces lividans

2002 ◽  
Vol 68 (10) ◽  
pp. 4731-4739 ◽  
Author(s):  
Michael J. Butler ◽  
Per Bruheim ◽  
Srdjan Jovetic ◽  
Flavia Marinelli ◽  
Pieter W. Postma ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Lividans ◽  
Pentose Phosphate ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
In The Wild ◽  
Commercially Important ◽  
Biochemical Product ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Wild Type Control

ABSTRACT Deletions were made in Streptomyces lividans in either of two genes (zwf1 and zwf2) encoding isozymes of glucose-6-phosphate dehydrogenase, the first enzyme in the oxidative pentose phosphate pathway (PPP). Each mutation reduced the level of Zwf activity to approximately one-half that observed in the wild-type strain. When the mutants were transformed with multicopy plasmids carrying the pathway-specific transcriptional activator genes for either the actinorhodin (ACT) or undecylprodigiosin (RED) biosynthetic pathway, they produced higher levels of antibiotic than the corresponding wild-type control strains. The presumed lower flux of carbon through the PPP in each of the Δzwf mutants may allow more efficient glucose utilization via glycolysis, resulting in higher levels of antibiotic production. This appears to occur without lowering the concentration of NADPH (the major biochemical product of the oxidative PPP activity) to a level that would limit antibiotic biosynthesis. Consistent with this hypothesis, deletion of the gene (devB) encoding the enzyme that catalyzes the next step in the oxidative PPP (6-phosphogluconolactonase) also resulted in increased antibiotic production. However, deletion of both zwf genes from the devB mutant resulted in reduced levels of ACT and RED production, suggesting that some of the NADPH made by the PPP is utilized, directly or indirectly, for antibiotic biosynthesis. Although applied here to the model antibiotics ACT and RED, such mutations may prove to be useful for improving the yield of commercially important secondary metabolites.

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.

scholarly journals The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

2004 ◽  
Vol 3 (3) ◽  
pp. 589-597 ◽  
Author(s):  
Michele Saliola ◽  
Paola Chiara Bartoccioni ◽  
Ilaria De Maria ◽  
Tiziana Lodi ◽  
Claudio Falcone
Keyword(s):  
Succinate Dehydrogenase ◽  
Glyoxylate Cycle ◽  
Alternative Pathway ◽  
Kluyveromyces Lactis ◽  
Carbon Sources ◽  
Pyruvate Decarboxylase ◽  
Northern Analysis ◽  
Gene Encoding ◽  
Dehydrogenase Gene ◽  
High Sequence Conservation

ABSTRACT We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose.

Effects of external phosphate concentration on glucose-6-phosphate dehydrogenase gene expression in the arbuscular mycorrhizal fungus Glomus intraradices

2006 ◽  
Vol 52 (9) ◽  
pp. 823-830 ◽  
Author(s):  
L I Stewart ◽  
S Jabaji-Hare ◽  
B T Driscoll
Keyword(s):  
Gene Expression ◽  
Mycorrhizal Fungi ◽  
Glomus Intraradices ◽  
Arbuscular Mycorrhizal Fungus ◽  
Mycorrhizal Fungus ◽  
Arbuscular Mycorrhizal ◽  
Pentose Phosphate ◽  
Dehydrogenase Gene ◽  
Significant Difference ◽  
Glucose 6 Phosphate Dehydrogenase

Specific primers were developed to amplify a 227 bp segment of the arbuscular mycorrhizal fungus Glomus intraradices gene encoding glucose-6-phosphate dehydrogenase (G6PDH), an enzyme involved in the pentose phosphate pathway. G6PDH gene expression was measured by real-time quantitative reverse transcriptase – polymerase chain reaction in response to phosphorus (P) concentrations in the growth medium of colonized transformed carrot roots. We investigated the effects of different P concentration treatments on carbon (C) metabolism within the intraradical mycelia of G. intraradices. The results showed a significant (P = 0.017) down-regulation of G6PDH expression in the intraradical mycelia of G. intraradices cultures grown in high P than low P conditions but no significant difference in regulation in excessive P concentrations when compared with the low P or high P concentrations. These results indicate that a reduction in the C flow from the host could be occurring as a result of elevated P and that a decrease in fungal G6PDH gene expression occurs, but not in the short term (less than 2 h). Reduced C flow from the host could lead to reduced fungal growth and root colonization, as was observed under high soil P conditions.Key words: arbuscular mycorrhizal fungi, phosphorus, nutrient uptake, glucose-6-phosphate dehydrogenase, gene expression.

scholarly journals Unique Cellular and Biochemical Features of Human Mitochondrial Peroxiredoxin 3 Establish the Molecular Basis for Its Specific Reaction with Thiostrepton

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 150
Author(s):  
Kimberly J. Nelson ◽  
Terri Messier ◽  
Stephanie Milczarek ◽  
Alexis Saaman ◽  
Stacie Beuschel ◽  
...  
Keyword(s):  
Oxygen Species ◽  
Mitochondrial Reactive Oxygen Species ◽  
Wild Type ◽  
Cellular Models ◽  
Chemotherapeutic Approach ◽  
Promising Target ◽  
Redox Responsive ◽  
Catalytic Cysteine ◽  
Increased Sensitivity ◽  
Peroxiredoxin 3

A central hallmark of tumorigenesis is metabolic alterations that increase mitochondrial reactive oxygen species (mROS). In response, cancer cells upregulate their antioxidant capacity and redox-responsive signaling pathways. A promising chemotherapeutic approach is to increase ROS to levels incompatible with tumor cell survival. Mitochondrial peroxiredoxin 3 (PRX3) plays a significant role in detoxifying hydrogen peroxide (H2O2). PRX3 is a molecular target of thiostrepton (TS), a natural product and FDA-approved antibiotic. TS inactivates PRX3 by covalently adducting its two catalytic cysteine residues and crosslinking the homodimer. Using cellular models of malignant mesothelioma, we show here that PRX3 expression and mROS levels in cells correlate with sensitivity to TS and that TS reacts selectively with PRX3 relative to other PRX isoforms. Using recombinant PRXs 1–5, we demonstrate that TS preferentially reacts with a reduced thiolate in the PRX3 dimer at mitochondrial pH. We also show that partially oxidized PRX3 fully dissociates to dimers, while partially oxidized PRX1 and PRX2 remain largely decameric. The ability of TS to react with engineered dimers of PRX1 and PRX2 at mitochondrial pH, but inefficiently with wild-type decameric protein at cytoplasmic pH, supports a novel mechanism of action and explains the specificity of TS for PRX3. Thus, the unique structure and propensity of PRX3 to form dimers contribute to its increased sensitivity to TS-mediated inactivation, making PRX3 a promising target for prooxidant cancer therapy.

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