ABSTRACT
Innovative strategies to control malaria are urgently needed. Exploring the interplay between Plasmodium sp. parasites and host red blood cells (RBCs) offers opportunities for novel antimalarial interventions. Pyruvate kinase deficiency (PKD), characterized by heightened 2,3-diphosphoglycerate (2,3-DPG) concentration, has been associated with protection against malaria. Elevated levels of 2,3-DPG, a specific mammalian metabolite, may hinder glycolysis, prompting us to hypothesize its potential contribution to PKD-mediated protection. We investigated the impact of the extracellular supplementation of 2,3-DPG on the Plasmodium falciparum intraerythrocytic developmental cycle in vitro. The results showed an inhibition of parasite growth, resulting from significantly fewer progeny from 2,3-DPG-treated parasites. We analyzed differential gene expression and the transcriptomic profile of P. falciparum trophozoites, from in vitro cultures subjected or not subjected to the action of 2,3-DPG, using Nanopore Sequencing Technology. The presence of 2,3-DPG in the culture medium was associated with the significant differential expression of 71 genes, mostly associated with the GO terms nucleic acid binding, transcription or monoatomic anion channel. Further, several genes related to cell cycle control were downregulated in treated parasites. These findings suggest that the presence of this RBC-specific glycolytic metabolite impacts the expression of genes transcribed during the parasite trophozoite stage and the number of merozoites released from individual schizonts, which supports the potential role of 2,3-DPG in the mechanism of protection against malaria by PKD.
Subject(s)
Malaria, Falciparum , Parasites , Animals , 2,3-Diphosphoglycerate/metabolism , Diphosphoglyceric Acids/metabolism , Malaria, Falciparum/genetics , Malaria, Falciparum/metabolism , Plasmodium falciparum/genetics , Glycolysis/genetics , Erythrocytes/metabolism , Gene Expression , MammalsABSTRACT
Malaria remains a major world public health problem, contributing to poverty and inequality. It is urgent to find new efficacious tools with few adverse effects. Malaria has selected red blood cell (RBC) alterations linked to resistance against infection, and understanding the protective mechanisms involved may be useful for developing host-directed tools to control Plasmodium infection. Pyruvate kinase deficiency has been associated with resistance to malaria. Pyruvate kinase-deficient RBCs display an increased concentration of 2,3-diphosphoglycerate (2,3-DPG). We recently showed that 2,3-DPG impacts in vitro intraerythrocytic parasite growth, induces a shift of the metabolic profile of infected cells (iRBCs), making it closer to that of noninfected ones (niRBCs), and decreases the number of parasite progenies that invade new RBCs. As an increase of 2,3-DPG content may also have an adverse effect on RBC membrane and, consequently, on the parasite invasion, in this study, we explored modifications of the RBC morphology, biomechanical properties, and RBC membrane on Plasmodium falciparum in vitro cultures treated with 2,3-DPG, using atomic force microscopy (AFM)-based force spectroscopy and other experimental approaches. The presence of infection by P. falciparum significantly increased the rigidity of parasitized cells and influenced the morphology of RBCs, as parasitized cells showed a decrease of the area-to-volume ratio. The extracellular addition of 2,3-DPG also slightly affected the stiffness of niRBCs, making it more similar to that of infected cells. It also changed the niRBC height, making the cells appear more elongated. Moreover, 2,3-DPG treatment influenced the cell surface charge, becoming more negative in treated RBCs than in untreated ones. The results indicate that treatment with 2,3-DPG has only a mild effect on RBCs in comparison with the effect of the presence of the parasite on the host cell. 2,3-DPG is an endogenous host metabolite, which may, in the future, originate a new antimalarial tool with few adverse effects on noninfected cells.
Subject(s)
Malaria, Falciparum , Malaria , Humans , 2,3-Diphosphoglycerate/metabolism , Pyruvate Kinase/metabolism , Erythrocytes/metabolism , Malaria/metabolism , Malaria, Falciparum/parasitology , Plasmodium falciparum , Diphosphoglyceric Acids/metabolismABSTRACT
The problem of analysis of clinical - diagnostic and biochemical criteria of postoperative cognitive dysfunction in abdominal oncosurgery, depending on the degree and structure of disorders, remains unresolved, which determines its relevance. The role of 2, 3-diphosphoglycerate is essential, because its increase in the concentration of red blood cells in hypoxic conditions is one of the adaptive mechanisms that improve oxygen transport to tissues. Purpose. The influence of the dynamics of 2,3 diphosphoglycerate content, as the main indicator of hypoxia, on the occurrence of cognitive dysfunction in the postoperative period in patients with neoplasms of the abdominal cavity. The study was conducted on the basis of departments for patients of the surgical profile of the municipal institution "Kharkiv City Clinical Hospital of Ambulance and Emergency Care named after Professor OI Meshchaninov ". To achieve this goal, we examined 80 patients with abdominal neoplasms who underwent surgery under general anesthesia using propofol and fentanyl. All patients were divided into 2 groups depending on the age of patients on the WHO scale, who underwent surgery using general anesthesia: Group 1 (n = 39) - middle-aged patients (50-59 years); Group 2 (n = 41) - elderly and senile patients (60-80 years). The control points of the examination were the day before the operation and the 1st, 7th, 30th day from the moment of the operation. The state of cognitive function in these patients was determined by conducting neuropsychological tests. To assess the state of cognitive function of patients, neuropsychological tests were used: MMSE scale (Mini-Mental State Examination, MMSE), the method of memorizing 10 words AR Luria, frontal dysfunction battery (FAB), Schulte technique. To assess the state of energy metabolism in patients, the level of erythrocytes and hemoglobin in the blood analysis was determined by well-known methods, the level of 2,3 diphosphoglycerate in erythrocytes and its ratio to hemoglobin. Anemia in the first week after surgery in patients of group 1 contributes to the development of a hypoxic state, in erythrocytes there is an increase in the content of 2,3 41 diphosphoglycerate, which promotes the transport of oxygen to tissues. During the week there is an increase in the intensity of the formation of 2,3 diphosphoglycerate, as evidenced by the ratio of 2,3 diphosphoglycerate to hemoglobin. In patients of group 2, the changes are more pronounced: anemia with a significant decrease in erythrocytes and hemoglobin in the blood, a decrease in 2.3 diphosphoglycerate in erythrocytes, reflects changes in erythrocyte metabolism, namely a decrease in biosynthesis of important organophosphorus compounds, in particular 2,3 diphosphogly by reducing the basic enzymes of glycolysis. Decreased energy metabolism in the elderly contributes to impaired cell function. With age, the content of adenosine triphosphate, 2,3 diphosphoglycerate decreases, thus increasing the affinity of hemoglobin for oxygen, impaired transport of oxygen to tissues, which leads to the development of hypoxia. According to the results of neuropsychological tests, we found postoperative cognitive dysfunction in patients with neoplasms of the abdominal cavity. Disruption of energy metabolism and changes in the activity of glycolysis enzymes in erythrocytes contributes to a decrease in the concentration of 2, 3 diphosphoglycerate, increase the affinity of hemoglobin for oxygen and the development of tissue hypoxia. The obtained results indicate the interdependence of these processes and allow continuing research in this direction with the development of appropriate clinical and diagnostic measures and areas of intensive care to improve the condition of patients with abdominal tumors and their quality of life after surgery.
Subject(s)
Abdominal Neoplasms , Anemia , Cognitive Dysfunction , Postoperative Cognitive Complications , 2,3-Diphosphoglycerate/metabolism , Abdominal Neoplasms/complications , Abdominal Neoplasms/metabolism , Abdominal Neoplasms/surgery , Aged , Cognitive Dysfunction/diagnosis , Cognitive Dysfunction/etiology , Diphosphoglyceric Acids/metabolism , Erythrocytes/metabolism , Hemoglobins , Humans , Hypoxia , Middle Aged , Oxygen/metabolism , Postoperative Period , Quality of LifeABSTRACT
BACKGROUND: Klebsiella pneumoniae is a bacterium that can be used as producer for numerous chemicals. Glycerol can be catabolised by K. pneumoniae and dihydroxyacetone is an intermediate of this catabolism pathway. Here dihydroxyacetone and glycerol were produced from glucose by this bacterium based a redirected glycerol catabolism pathway. RESULTS: tpiA, encoding triosephosphate isomerase, was knocked out to block the further catabolism of dihydroxyacetone phosphate in the glycolysis. After overexpression of a Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase (hdpA), the engineered strain produced remarkable levels of dihydroxyacetone (7.0 g/L) and glycerol (2.5 g/L) from glucose. Further increase in product formation were obtained by knocking out gapA encoding an iosenzyme of glyceraldehyde 3-phosphate dehydrogenase. There are two dihydroxyacetone kinases in K. pneumoniae. They were both disrupted to prevent an inefficient reaction cycle between dihydroxyacetone phosphate and dihydroxyacetone, and the resulting strains had a distinct improvement in dihydroxyacetone and glycerol production. pH 6.0 and low air supplement were identified as the optimal conditions for dihydroxyacetone and glycerol production by K, pneumoniae ΔtpiA-ΔDHAK-hdpA. In fed batch fermentation 23.9 g/L of dihydroxyacetone and 10.8 g/L of glycerol were produced after 91 h of cultivation, with the total conversion ratio of 0.97 mol/mol glucose. CONCLUSIONS: This study provides a novel and highly efficient way of dihydroxyacetone and glycerol production from glucose.
Subject(s)
Dihydroxyacetone/metabolism , Klebsiella pneumoniae/metabolism , Dihydroxyacetone Phosphate/metabolism , Diphosphoglyceric Acids/metabolism , Fermentation , Genes, Bacterial , Glucose/metabolism , Glyceraldehyde 3-Phosphate/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Glycerol/metabolism , Hydrogen-Ion Concentration , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/growth & development , Metabolic Engineering , Metabolic Networks and Pathways , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , ThermodynamicsABSTRACT
OBJECTIVE: The pathogenesis of IgG4-related disease (IgG4-RD) remains unclear. Metabolomic profiling of IgG4-RD patients offers an opportunity to identify novel pathophysiological targets and biomarkers. This study aims to identify potential plasma biomarkers associated with IgG4-RD. METHODS: Thirty newly diagnosed IgG4-RD patients, age-matched healthy controls and post-treated IgG4-RD patients were enrolled. Patients' clinical data, laboratory parameters and plasma were collected. Plasma was measured for ultraperformance liquid chromatography-tandem mass spectrometry based metabolomics and lipidomics profiling. Multivariate and univariate statistical analyses were conducted to identify potential biomarkers. The receiver operating characteristic and the correlations between biomarkers and clinical parameters were investigated. RESULTS: The plasma metabolites are altered among healthy controls, newly diagnosed IgG4-RD and post-treated IgG4-RD groups. Of the identified features, eight metabolites were significantly perturbed in the IgG4-RD group, including glyceric acid 1,3-biphosphate (1,3-BPG), uridine triphosphate (UTP), uridine diphosphate glucose (UDP-Glc) or uridine diphosphate galactose (UDP-Gal), lysophospholipids, linoleic acid derivatives and ceramides. Receiver operating characteristic analysis indicated that UTP, UDP-Glc/UDP-Gal and LysoPC (18:1) had high sensitivity and specificity in diagnosis of IgG4-RD. A Pearson correlation analysis showed that 1,3-BPG and UTP were strongly correlated with clinical parameters. CONCLUSION: IgG4-RD patients have a unique plasma metabolomic profile compared with healthy controls. Our study suggested that metabolomic profiling may provide important insights into pathophysiology and testable biomarkers for diagnosis of IgG4-RD.
Subject(s)
Immunoglobulin G4-Related Disease/metabolism , Lipidomics , Metabolomics , Adult , Case-Control Studies , Ceramides/metabolism , Chromatography, Liquid , Diphosphoglyceric Acids/metabolism , Female , Humans , Linoleic Acids/metabolism , Lysophospholipids/metabolism , Male , Middle Aged , Tandem Mass Spectrometry , Uridine Diphosphate Galactose/metabolism , Uridine Diphosphate Glucose/metabolism , Uridine Triphosphate/metabolismABSTRACT
Chemicals inducing a mild decrease in the ATP/ADP ratio are considered as caloric restriction mimetics as well as treatments against obesity. Screening for such chemicals in animal model systems requires a lot of time and labor. Here, we present a system for the rapid screening of non-toxic substances causing such a de-energization of cells. We looked for chemicals allowing the growth of yeast lacking trehalose phosphate synthase on a non-fermentable carbon source in the presence of glucose. Under such conditions, the cells cannot grow because the cellular phosphate is mostly being used to phosphorylate the sugars in upper glycolysis, while the biosynthesis of bisphosphoglycerate is blocked. We reasoned that by decreasing the ATP/ADP ratio, one might prevent the phosphorylation of the sugars and also boost bisphosphoglycerate synthesis by providing the substrate, i.e., inorganic phosphate. We confirmed that a complete inhibition of oxidative phosphorylation alleviates the block. As our system includes a non-fermentable carbon source, only the chemicals that did not cause a complete block of mitochondrial ATP synthesis allowed the initial depletion of glucose followed by respiratory growth. Using this system, we found two novel compounds, dodecylmethyl diphenylamine (FS1) and diethyl (tetradecyl) phenyl ammonium bromide (Kor105), which possess a mild membrane-depolarizing activity.
Subject(s)
Adenosine Triphosphate/metabolism , Cations/metabolism , Glucose/metabolism , Glucosyltransferases/metabolism , Glycolysis , Saccharomyces cerevisiae/metabolism , Adenosine Diphosphate/metabolism , Animals , Diphosphoglyceric Acids/metabolism , Glucosyltransferases/genetics , Mitochondria/metabolism , Mitochondria, Liver/metabolism , Models, Biological , Oxidative Phosphorylation , Oxygen Consumption , Phosphates/metabolism , Rats , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & developmentABSTRACT
Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The Km values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 µm (yeast PGK1) and 6.86 µm (human PKG1). The Km values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 µm (yeast PGK1) and 186 µm (human PGK1). The Vmax of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 µm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis.
Subject(s)
Glycolysis , Neoplasm Proteins , Neoplasms , Phosphoglycerate Kinase , A549 Cells , Diphosphoglyceric Acids/chemistry , Diphosphoglyceric Acids/metabolism , Glucose/chemistry , Glucose/metabolism , Glyceric Acids/chemistry , Glyceric Acids/metabolism , HeLa Cells , Humans , Kinetics , Lactic Acid/chemistry , Lactic Acid/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Neoplasms/chemistry , Neoplasms/metabolism , Phosphoglycerate Kinase/chemistry , Phosphoglycerate Kinase/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
BACKGROUND: Post-translational modification (PTM), which is a biological process, tends to modify proteome that leads to changes in normal cell biology and pathogenesis. In the recent times, there has been many reported PTMs. Out of the many modifications, phosphoglycerylation has become particularly the subject of interest. The experimental procedure for identification of phosphoglycerylated residues continues to be an expensive, inefficient and time-consuming effort, even with a large number of proteins that are sequenced in the post-genomic period. Computational methods are therefore being anticipated in order to effectively predict phosphoglycerylated lysines. Even though there are predictors available, the ability to detect phosphoglycerylated lysine residues still remains inadequate. RESULTS: We have introduced a new predictor in this paper named EvolStruct-Phogly that uses structural and evolutionary information relating to amino acids to predict phosphoglycerylated lysine residues. Benchmarked data is employed containing experimentally identified phosphoglycerylated and non-phosphoglycerylated lysines. We have then extracted the three structural information which are accessible surface area of amino acids, backbone torsion angles, amino acid's local structure conformations and profile bigrams of position-specific scoring matrices. CONCLUSION: EvolStruct-Phogly showed a noteworthy improvement in regards to the performance when compared with the previous predictors. The performance metrics obtained are as follows: sensitivity 0.7744, specificity 0.8533, precision 0.7368, accuracy 0.8275, and Mathews correlation coefficient of 0.6242. The software package and data of this work can be obtained from https://github.com/abelavit/EvolStruct-Phogly or www.alok-ai-lab.com.
Subject(s)
Computational Biology/methods , Diphosphoglyceric Acids/chemistry , Evolution, Molecular , Protein Processing, Post-Translational , Proteins/chemistry , Algorithms , Binding Sites , Diphosphoglyceric Acids/metabolism , Humans , Lysine/chemistry , Lysine/metabolism , Proteins/metabolism , Sequence Analysis, Protein , Software , Support Vector MachineABSTRACT
Post-translational modification (PTM) is essential for many biological processes. Covalent and generally enzymatic modification of proteins can impact the activity of proteins. Modified proteins would have more complex structures and functions. Knowing whether a specific residue is modified or not is significant to unravel the function and structure of this protein. As experimental approaches to discover protein PTM sites are always costly and time consuming, computational prediction methods are desirable alternative methods. Lysine phosphoglycerylation is a type of newly discovered PTM that is related to glycolytic process and glucose metabolism. Since the lysine phosphoglycerylation process requires no catalytic enzyme, its site selectivity mechanism is still not fully understood. In this study, we designed a novel computational method, namely PhoglyPred, to identify lysine phosphoglycerylation sites. By utilizing several different protein sequence descriptors, PhoglyPred achieved an overall accuracy of 90.3% in a Jackknife test, which is better than other state-of-the-art predictors. By analyzing the importance of different features using the F-score, we found several important sequence features, which may benefit future studies in understanding the site selectivity mechanism of lysine phosphoglycerylation.
Subject(s)
Computational Biology/methods , Diphosphoglyceric Acids/metabolism , Lysine/metabolism , Proteins/chemistry , Algorithms , Binding Sites , Glucose/metabolism , Glycolysis , Protein Processing, Post-Translational , Support Vector MachineABSTRACT
Metabolomic profiling studies aim to provide a comprehensive, quantitative, and dynamic portrait of the endogenous metabolites in a biological system. While contemporary technologies permit routine profiling of many metabolites, intrinsically labile metabolites are often improperly measured or omitted from studies due to unwanted chemical transformations that occur during sample preparation or mass spectrometric analysis. The primary glycolytic metabolite 1,3-bisphosphoglyceric acid (1,3-BPG) typifies this class of metabolites, and, despite its central position in metabolism, has largely eluded analysis in profiling studies. Here we take advantage of the reactive acylphosphate group in 1,3-BPG to chemically trap the metabolite with hydroxylamine during metabolite isolation, enabling quantitative analysis by targeted LC-MS/MS. This approach is compatible with complex cellular metabolome, permits specific detection of the reactive (1,3-) instead of nonreactive (2,3-) BPG isomer, and has enabled direct analysis of dynamic 1,3-BPG levels resulting from perturbations to glucose processing. These studies confirmed that standard metabolomic methods misrepresent cellular 1,3-BPG levels in response to altered glucose metabolism and underscore the potential for chemical trapping to be used for other classes of reactive metabolites.
Subject(s)
Diphosphoglyceric Acids/chemistry , Glucose/metabolism , Hydroxylamine/chemistry , Metabolome , Tandem Mass Spectrometry , Cell Line, Tumor , Chromatography, High Pressure Liquid , Diphosphoglyceric Acids/metabolism , Glucose/chemistry , Humans , IsomerismABSTRACT
As a new type of post-translational modification, lysine phosphoglycerylation plays a key role in regulating glycolytic process and metabolism in cells. Due to the traditional experimental methods are time-consuming and labor-intensive, it is important to develop computational methods to identify the potential phosphoglycerylation sites. However, the prediction performance of the existing phosphoglycerylation site predictor is not satisfactory. In this study, a novel predictor named CKSAAP_PhoglySite is developed to predict phosphoglycerylation sites by using composition of k-spaced amino acid pairs and fuzzy support vector machine. On the one hand, after many aspects of assessments, we find the composition of k-spaced amino acid pairs is more suitable for representing the protein sequence around the phosphoglycerylation sites than other encoding schemes. On the other hand, the proposed fuzzy support vector machine algorithm can effectively handle the imbalanced and noisy problem in phosphoglycerylation sites training dataset. Experimental results indicate that CKSAAP_PhoglySite outperforms the existing phosphoglycerylation site predictor Phogly-PseAAC significantly. A matlab software package for CKSAAP_PhoglySite can be freely downloaded from https://github.com/juzhe1120/Matlab_Software/blob/master/CKSAAP_PhoglySite_Matlab_Software.zip.
Subject(s)
Algorithms , Amino Acids/metabolism , Computational Biology/methods , Fuzzy Logic , Lysine/metabolism , Support Vector Machine , Amino Acid Sequence , Amino Acids/genetics , Binding Sites/genetics , Diphosphoglyceric Acids/metabolism , Lysine/genetics , Protein Processing, Post-Translational , Reproducibility of Results , SoftwareABSTRACT
Cancer cells exhibit profound metabolic alterations, allowing them to fulfill the metabolic needs that come with increased proliferation and additional facets of malignancy. Such a metabolic transformation is orchestrated by the genetic changes that drive tumorigenesis, that is, the activation of oncogenes and/or the loss of oncosuppressor genes, and further shaped by environmental cues, such as oxygen concentration and nutrient availability. Understanding this metabolic rewiring is essential to elucidate the fundamental mechanisms of tumorigenesis as well as to find novel, therapeutically exploitable liabilities of malignant cells. Here, we describe key features of the metabolic transformation of cancer cells, which frequently include the switch to aerobic glycolysis, a profound mitochondrial reprogramming, and the deregulation of lipid metabolism, highlighting the notion that these pathways are not independent but rather cooperate to sustain proliferation. Finally, we hypothesize that only those genetic defects that effectively support anabolism are selected in the course of tumor progression, implying that cancer-associated mutations may undergo a metabolically convergent evolution.
Subject(s)
Glycolysis , Mitochondria/metabolism , Neoplasms/metabolism , Cell Transformation, Neoplastic , Diphosphoglyceric Acids/metabolism , Fatty Acids/biosynthesis , Humans , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Lipid Metabolism , Mutation , Neoplasms/genetics , Oncogenes , Phosphofructokinase-1/metabolism , Phosphofructokinase-2/metabolism , Phosphoglycerate Dehydrogenase/metabolism , Phosphoglycerate Mutase/metabolism , Pyruvate Kinase/metabolismABSTRACT
BACKGROUND: Corynebacterium glutamicum cg1790/pgk encodes an enzyme active as a 3-phosphoglycerate kinase (PGK) (EC 2.7.2.3) catalyzing phosphoryl transfer from 1,3-biphosphoglycerate (bPG) to ADP to yield 3-phosphoglycerate (3-PG) and ATP in substrate chain phosphorylation. RESULTS: C. glutamicum 3-phosphoglycerate kinase was purified to homogeneity from the soluble fraction of recombinant E. coli. PGK(His) was found to be active as a homodimer with molecular weight of 104 kDa. The enzyme preferred conditions of pH 7.0 to 7.4 and required Mg²âº for its activity. PGK(His) is thermo labile and it has shown maximal activity at 50-65°C. The maximal activity of PGK(His) was estimated to be 220 and 150 U mg-1 with KM values of 0.26 and 0.11 mM for 3-phosphoglycerate and ATP, respectively. A 3-phosphoglycerate kinase negative C. glutamicum strain ∆pgk was constructed and shown to lack the ability to grow under glycolytic or gluconeogenic conditions unless PGK was expressed from a plasmid to restore growth. When pgk was overexpressed in L-arginine and L-ornithine production strains the production increased by 8% and by 17.5%, respectively. CONCLUSION: Unlike many bacterial PGKs, C. glutamicum PGK is active as a homodimer. PGK is essential for growth of C. glutamicum with carbon sources requiring glycolysis and gluconeogenesis. Competitive inhibition by ADP reveals the critical role of PGK in gluconeogenesis by energy charge. Pgk overexpression improved the productivity in L-arginine and L-ornithine production strains.
Subject(s)
Amino Acids/metabolism , Corynebacterium glutamicum/enzymology , Phosphoglycerate Kinase/genetics , Phosphoglycerate Kinase/metabolism , Adenosine Diphosphate/metabolism , Coenzymes/metabolism , Corynebacterium glutamicum/genetics , Corynebacterium glutamicum/growth & development , Corynebacterium glutamicum/metabolism , Diphosphoglyceric Acids/metabolism , Enzyme Stability , Escherichia coli/genetics , Gene Deletion , Glycolysis , Hydrogen-Ion Concentration , Kinetics , Magnesium/metabolism , Molecular Weight , Phosphoglycerate Kinase/chemistry , Phosphoglycerate Kinase/isolation & purification , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
BACKGROUND: Previously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production. METHODOLOGY/PRINCIPAL FINDINGS: In the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD(+) to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD(+). In this study, to improve 2,3-BD production, we first over-produced NAD(+)-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h. CONCLUSIONS/SIGNIFICANCE: Co-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms.
Subject(s)
Alcohol Oxidoreductases/genetics , Bacillus/genetics , Bacillus/metabolism , Butylene Glycols/metabolism , Gene Expression , Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)/genetics , Alcohol Oxidoreductases/metabolism , Batch Cell Culture Techniques , Bioreactors , Diphosphoglyceric Acids/metabolism , Fermentation , Gene Order , Genetic Vectors/genetics , Glucose/metabolism , Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)/metabolism , Metabolic Networks and PathwaysABSTRACT
The posttranslational modification of proteins and their regulation by metabolites represent conserved mechanisms in biology. At the confluence of these two processes, we report that the primary glycolytic intermediate 1,3-bisphosphoglycerate (1,3-BPG) reacts with select lysine residues in proteins to form 3-phosphoglyceryl-lysine (pgK). This reaction, which does not require enzyme catalysis, but rather exploits the electrophilicity of 1,3-BPG, was found by proteomic profiling to be enriched on diverse classes of proteins and prominently in or around the active sites of glycolytic enzymes. pgK modifications inhibit glycolytic enzymes and, in cells exposed to high glucose, accumulate on these enzymes to create a potential feedback mechanism that contributes to the buildup and redirection of glycolytic intermediates to alternate biosynthetic pathways.
Subject(s)
Diphosphoglyceric Acids/metabolism , Glycerophosphates/metabolism , Glycolysis , Lysine/analogs & derivatives , Lysine/metabolism , Protein Processing, Post-Translational , Proteins/metabolism , Amino Acid Sequence , Animals , Biomarkers, Tumor/chemistry , Biomarkers, Tumor/metabolism , Catalysis , Cell Line , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Glucose/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/chemistry , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism , Humans , Mice , Molecular Sequence Data , Phosphopyruvate Hydratase/chemistry , Phosphopyruvate Hydratase/metabolism , Proteins/chemistry , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/metabolismABSTRACT
Four enzymes of the gluconeogenic pathway in Sulfolobus solfataricus were purified and kinetically characterized. The enzymes were reconstituted in vitro to quantify the contribution of temperature instability of the pathway intermediates to carbon loss from the system. The reconstituted system, consisting of phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase and the fructose 1,6-bisphosphate aldolase/phosphatase, maintained a constant consumption rate of 3-phosphoglycerate and production of fructose 6-phosphate over a 1-h period. Cofactors ATP and NADPH were regenerated via pyruvate kinase and glucose dehydrogenase. A mathematical model was constructed on the basis of the kinetics of the purified enzymes and the measured half-life times of the pathway intermediates. The model quantitatively predicted the system fluxes and metabolite concentrations. Relative enzyme concentrations were chosen such that half the carbon in the system was lost due to degradation of the thermolabile intermediates dihydroxyacetone phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate, indicating that intermediate instability at high temperature can significantly affect pathway efficiency.
Subject(s)
Archaeal Proteins/metabolism , Fructose-Bisphosphate Aldolase/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Models, Statistical , Phosphoglycerate Kinase/metabolism , Sulfolobus solfataricus/enzymology , Triose-Phosphate Isomerase/metabolism , Archaeal Proteins/genetics , Dihydroxyacetone Phosphate/metabolism , Diphosphoglyceric Acids/metabolism , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Fructose-Bisphosphate Aldolase/genetics , Fructosephosphates/biosynthesis , Gluconeogenesis/genetics , Glyceraldehyde 3-Phosphate/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glyceric Acids/metabolism , Half-Life , Hot Temperature , Kinetics , Phosphoglycerate Kinase/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sulfolobus solfataricus/chemistry , Sulfolobus solfataricus/genetics , Thermodynamics , Triose-Phosphate Isomerase/geneticsABSTRACT
A stochastic model for the dynamics of enzymatic catalysis in explicit, effective solvents under physiological conditions is presented. Analytically-computed first passage time densities of a diffusing particle in a spherical shell with absorbing boundaries are combined with densities obtained from explicit simulation to obtain the overall probability density for the total reaction cycle time of the enzymatic system. The method is used to investigate the catalytic transfer of a phosphoryl group in a phosphoglycerate kinase-ADP-bis phosphoglycerate system, one of the steps of glycolysis. The direct simulation of the enzyme-substrate binding and reaction is carried out using an elastic network model for the protein, and the solvent motions are described by multiparticle collision dynamics which incorporates hydrodynamic flow effects. Systems where solvent-enzyme coupling occurs through explicit intermolecular interactions, as well as systems where this coupling is taken into account by including the protein and substrate in the multiparticle collision step, are investigated and compared with simulations where hydrodynamic coupling is absent. It is demonstrated that the flow of solvent particles around the enzyme facilitates the large-scale hinge motion of the enzyme with bound substrates, and has a significant impact on the shape of the probability densities and average time scales of substrate binding for substrates near the enzyme, the closure of the enzyme after binding, and the overall time of completion of the cycle.
Subject(s)
Biocatalysis , Models, Biological , Phosphoglycerate Kinase/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Computer Simulation , Diffusion , Diphosphoglyceric Acids/metabolism , Glyceric Acids/metabolism , Kinetics , Models, Molecular , Phosphoglycerate Kinase/chemistry , Protein Conformation , Solvents/metabolism , Stochastic ProcessesABSTRACT
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an essential role in glycolysis by catalyzing the conversion of D-glyceraldehyde 3-phosphate (D-G3P) to 1,3-diphosphoglycerate using NAD(+) as a cofactor. In this report, the GAPDH gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (GAPDH-tk) was cloned and the protein was purified to homogeneity. GAPDH-tk exists as a homotetramer with a native molecular mass of 145 kDa; the subunit molecular mass was 37 kDa. GAPDH-tk is a thermostable protein with a half-life of 5 h at 80-90°C. The apparent K (m) values for NAD(+) and D-G3P were 77.8 ± 7.5 µM and 49.3 ± 3.0 µM, respectively, with V (max) values of 45.1 ± 0.8 U/mg and 59.6 ± 1.3 U/mg, respectively. Transmission electron microscopy (TEM) and image processing confirmed that GAPDH-tk has a tetrameric structure. Interestingly, GAPDH-tk migrates as high molecular mass forms (~232 kDa and ~669 kDa) in response to oxidative stress.
Subject(s)
Archaeal Proteins/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Thermococcus/enzymology , Amino Acid Sequence , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Archaeal Proteins/isolation & purification , Catalytic Domain , Cloning, Molecular , Diphosphoglyceric Acids/metabolism , Enzyme Stability , Glyceraldehyde 3-Phosphate/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/isolation & purification , Half-Life , Hot Temperature , Kinetics , Microscopy, Electron, Transmission , Molecular Sequence Data , Molecular Weight , Mutation , NAD/metabolism , Oxidative Stress , Protein Structure, Quaternary , Structure-Activity Relationship , Thermococcus/geneticsABSTRACT
3-Phosphogycerate kinase (PGK) is a two domain enzyme, with a binding site of the 1,3-bisphosphoglycerate on the N-domain and of the ADP on the C-domain. To transfer a phosphate group the enzyme has to undergo a hinge bending motion from open to closed conformation to bring the substrates to close proximity. Molecular dynamics simulation was used to elucidate the effect of ligand binding onto the domain motions of this enzyme. The simulation results of the apo form indicate a hinge bending motion in the ns timescale while the time period of the hinge bending motion of the complex form is clearly over the 20 ns simulation time. The apo form exhibits several hinge points that contribute to the hinge bending motion while upon binding the ligands, the hinge bending becomes strictly restrained with one dominant hinge point in the vicinity of the substrates. At the same time, ligand binding results in an enhanced correlation of internal domain motions.