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Jacky L Snoep - One of the best experts on this subject based on the ideXlab platform.
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intermediate instability at high temperature leads to low pathway efficiency for an in vitro reconstituted system of gluconeogenesis in sulfolobus solfataricus
FEBS Journal, 2013Co-Authors: Theresa Kouril, Dominik Esser, Julia Christin Kort, Hans V Westerhoff, Bettina Siebers, Jacky L SnoepAbstract: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.
Bianca Maria Ranzi - One of the best experts on this subject based on the ideXlab platform.
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Alterations of the glucose metabolism in a triose phosphate Isomerase-negative Saccharomyces cerevisiae mutant.
Yeast (Chichester England), 2001Co-Authors: Concetta Compagno, Luca Brambilla, Daniele Capitanio, Francesco Boschi, Bianca Maria Ranzi, Danilo PorroAbstract:The absence of triose phosphate Isomerase activity causes an accumulation of only one of the two trioses, dihydroxyacetone phosphate, and this produces a shift in the final product of glucose catabolism from ethanol to glycerol (Compagno et al., 1996). Alterations of glucose metabolism imposed by the deletion of the TPI1 gene in Saccharomyces cerevisiae were studied in batch and continuous cultures. The Δtpi1 null mutant was unable to grow on glucose as the sole carbon source. The addition of ethanol or acetate in media containing glucose, but also raffinose or galactose, relieved this effect in batch cultivation, suggesting that the Crabtree effect is not the primary cause for the mutant's impaired growth on glucose. The addition of an energy source like formic acid restored glucose utilization, suggesting that a NADH/energy shortage in the Δtpi1 mutant could be a cause of the impaired growth on glucose. The amount of glycerol production in the Δtpi1 mutant could represent a good indicator of the fraction of carbon source channelled through glycolysis. Data obtained in continuous cultures on mixed substrates indicated that different contributions of glycolysis and gluconeogenesis, as well as of the HMP pathway, to glucose utilization by the Δtpi1 mutant may occur in relation to the fraction of ethanol present in the media. Copyright © 2001 John Wiley & Sons, Ltd.
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Alterations of the glucose metabolism in a triose phosphate Isomerase-negative Saccharomyces cerevisiae mutant
'Wiley', 2001Co-Authors: Concetta Compagno, Luca Brambilla, Daniele Capitanio, Francesco Boschi, Bianca Maria Ranzi, Danilo PorroAbstract:The absence of triose phosphate Isomerase activity causes an accumulation of only one of the two trioses, dihydroxyacetone phosphate, and this produces a shift in the final product of glucose catabolism from ethanol to glycerol (Compagno et al., 1996), Alterations of glucose metabolism imposed by the deletion of the TPI1 gene in Saccharomyces,cerevisiae were studied in batch and continuous cultures. The Delta tpi1 null mutant was unable to grow on glucose as the sole carbon source. The addition of ethanol or acetate in media containing glucose, but also raffinose or galactose, relieved this effect in batch cultivation, suggesting that the Crabtree effect is not the primary cause for the mutant's impaired growth on glucose. The addition of an energy source like formic acid restored glucose utilization, suggesting that a NADH/energy shortage in the Delta tpi1 mutant could be a cause of the impaired growth on glucose. The amount of glycerol production in the Delta tpi1 mutant could represent a good indicator of the fraction of carbon source channelled through glycolysis. Data obtained in continuous cultures on mixed substrates indicated that different contributions of glycolysis and gluconeogenesis, as well as of the HMP pathway, to glucose utilization by the Delta tpi1 mutant may occur in relation to the fraction of ethanol present in the media
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isolation nucleotide sequence and physiological relevance of the gene encoding triose phosphate Isomerase from kluyveromyces lactis
Applied and Environmental Microbiology, 1999Co-Authors: Concetta Compagno, Francesco Boschi, Danilo Porro, Agnese Daleffe, Bianca Maria RanziAbstract:Lack of triose phosphate Isomerase activity (TIM) is of special interest because this enzyme works at an important branch point of glycolytic flux. In this paper, we report the cloning and sequencing of the Kluyveromyces lactis gene encoding TIM. Unlike Saccharomyces cerevisiae ΔTPI1 mutants, the K. lactis mutant strain was found to be able to grow on glucose. Preliminary bioconversion experiments indicated that, like the S. cerevisiae TIM-deficient strain, the K. lactis TIM-deficient strain is able to produce glycerol with high yield.
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glycerol production in a triose phosphate Isomerase deficient mutant of saccharomyces cerevisiae
Biotechnology Progress, 1996Co-Authors: Concetta Compagno, Francesco Boschi, Bianca Maria RanziAbstract:Interesting challenges from metabolically engineered Saccharomyces cerevisiae cells arise from the opportunity to obtain yeast strains useful for the production of chemicals. In this paper, we show that engineered yeast cells deficient in the triose phosphate Isomerase activity are able to produce glycerol without the use of steering agents. High yields of conversion of glucose into glycerol (80-90% of the theoretical yield) and productivity (1.5 g L-1 h-1) have been obtained by a bioconversion process carried out in a poor and clean medium. We obtained indications that the growth phase at which the biomass was collected affect the process. The best results were obtained using cells collected at the end of exponential phase of growth. In perspective, the strategies and the information about the physiology of the cells described here could be useful for the developing of new biotechnological processes for glycerol production, outflanking the problems related to the use of high level of steering agents.
Derek T Logan - One of the best experts on this subject based on the ideXlab platform.
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structural and functional analyses of beta glucosidase 3b from thermotoga neapolitana a thermostable three domain representative of glycoside hydrolase 3
Journal of Molecular Biology, 2010Co-Authors: Tania Pozzo, Javier Linares Pasten, Eva Nordberg Karlsson, Derek T LoganAbstract:Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) family 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized-that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable beta-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 A. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 A resolution. Bgl3B from Th. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (alpha/beta)(8) domain similar to a triose phosphate Isomerase barrel, a five-stranded alpha/beta sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second beta-strand of the triose phosphate Isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the -1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the -1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small substrates, although it may be involved in the anchoring of the enzyme on large polymeric substrates and in thermostability.
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structural and functional analyses of beta glucosidase 3b from thermotoga neapolitana a thermostable three domain representative of glycoside hydrolase 3
Journal of Molecular Biology, 2010Co-Authors: Tania Pozzo, Javier Linares Pasten, Eva Nordberg Karlsson, Derek T LoganAbstract:Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized-that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable beta-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 A. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 A resolution. Bgl3B from Thermot. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (alpha/beta)(8) domain similar to a triose phosphate Isomerase barrel, a five-stranded alpha/beta sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second beta-strand of the triose phosphate Isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the -1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the -1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small substrates, although it may be involved in the anchoring of the enzyme on large polymeric substrates and in thermostability. (Less)
Rik K Wierenga - One of the best experts on this subject based on the ideXlab platform.
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the ionization of a buried glutamic acid is thermodynamically linked to the stability of leishmania mexicana triose phosphate Isomerase
FEBS Journal, 2000Co-Authors: Annemarie Lambeir, Jan Backmann, Javier Ruizsanz, V V Filimonov, Jens Erik Nielsen, Inari Kursula, B V Norledge, Rik K WierengaAbstract:The amino acid sequence of Leishmania mexicana triose phosphate Isomerase is unique in having at position 65 a glutamic acid instead of a glutamine. The stability properties of LmTIM and the E65Q mutant were investigated by pH and guanidinium chloride-induced unfolding. The crystal structure of E65Q was determined. Three important observations were made: (a) there are no structural rearrangements as the result of the substitution; (b) the mutant is more stable than the wild-type; and (c) the stability of the wild-type enzyme shows strong pH dependence, which can be attributed to the ionization of Glu65. Burying of the Glu65 side chain in the uncharged environment of the dimer interface results in a shift in pKa of more than 3 units. The pH-dependent decrease in overall stability is due to weakening of the monomer-monomer interactions (in the dimer). The E65Q substitution causes an increase in stability as the result of the formation of an additional hydrogen bond in each subunit (DeltaDeltaG degrees of 2 kcal.mol-1 per monomer) and the elimination of a charged group in the dimer interface (DeltaDeltaG degrees of at least 9 kcal.mol-1 per dimer). The computated shift in pKa and the stability of the dimer calculated from the charge distribution in the protein structure agree closely with the experimental results. The guanidinium chloride dependence of the unfolding constant was smaller than expected from studies involving monomeric model proteins. No intermediates could be identified in the unfolding equilibrium by combining fluorescence and CD measurements. Study of a stable monomeric triose phosphate Isomerase variant confirmed that the phenomenon persists in the monomer.
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triose phosphate Isomerase tim of the psychrophilic bacterium vibrio marinus kinetic and structural properties
Journal of Biological Chemistry, 1998Co-Authors: Marco Alvarez, Rik K Wierenga, J P Zeelen, Veronique Mainfroid, Francoise Rentierdelrue, Joseph Martial, Lode Wyns, Dominique MaesAbstract:Abstract The purification and characterization of Triose-Phosphate Isomerase from the psychrophilic bacteriumVibrio marinus (vTIM) is described. Crystal structures of the vTIM-sulfate complex and the vTIM-2-phosphoglycolate complex (at a 2.7-A resolution) are also presented. The optimal growth temperature of Vibrio marinus is 15 °C. Stability studies show that vTIM is an unstable protein with a half-life of only 10 min at 25 °C. The vTIM sequence is most closely related to the sequence ofEscherichia coli TIM (eTIM) (66% identity), and several unique structural features described for eTIM are also seen in vTIM, but eTIM is considerably more stable. The T d values of vTIM and eTIM, determined by calorimetric studies, are 41 and 54 °C, respectively. Amino acid sequence comparison reveals that vTIM has an alanine in loop 8 (at position 238), whereas all other TIM sequences known to date have a serine. The vTIM mutant A238S was produced and characterized. Compared with wild type, the catalytic efficiency of the A238S mutant is somewhat reduced, and its stability is considerably increased.
Concetta Compagno - One of the best experts on this subject based on the ideXlab platform.
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Alterations of the glucose metabolism in a triose phosphate Isomerase-negative Saccharomyces cerevisiae mutant.
Yeast (Chichester England), 2001Co-Authors: Concetta Compagno, Luca Brambilla, Daniele Capitanio, Francesco Boschi, Bianca Maria Ranzi, Danilo PorroAbstract:The absence of triose phosphate Isomerase activity causes an accumulation of only one of the two trioses, dihydroxyacetone phosphate, and this produces a shift in the final product of glucose catabolism from ethanol to glycerol (Compagno et al., 1996). Alterations of glucose metabolism imposed by the deletion of the TPI1 gene in Saccharomyces cerevisiae were studied in batch and continuous cultures. The Δtpi1 null mutant was unable to grow on glucose as the sole carbon source. The addition of ethanol or acetate in media containing glucose, but also raffinose or galactose, relieved this effect in batch cultivation, suggesting that the Crabtree effect is not the primary cause for the mutant's impaired growth on glucose. The addition of an energy source like formic acid restored glucose utilization, suggesting that a NADH/energy shortage in the Δtpi1 mutant could be a cause of the impaired growth on glucose. The amount of glycerol production in the Δtpi1 mutant could represent a good indicator of the fraction of carbon source channelled through glycolysis. Data obtained in continuous cultures on mixed substrates indicated that different contributions of glycolysis and gluconeogenesis, as well as of the HMP pathway, to glucose utilization by the Δtpi1 mutant may occur in relation to the fraction of ethanol present in the media. Copyright © 2001 John Wiley & Sons, Ltd.
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Alterations of the glucose metabolism in a triose phosphate Isomerase-negative Saccharomyces cerevisiae mutant
'Wiley', 2001Co-Authors: Concetta Compagno, Luca Brambilla, Daniele Capitanio, Francesco Boschi, Bianca Maria Ranzi, Danilo PorroAbstract:The absence of triose phosphate Isomerase activity causes an accumulation of only one of the two trioses, dihydroxyacetone phosphate, and this produces a shift in the final product of glucose catabolism from ethanol to glycerol (Compagno et al., 1996), Alterations of glucose metabolism imposed by the deletion of the TPI1 gene in Saccharomyces,cerevisiae were studied in batch and continuous cultures. The Delta tpi1 null mutant was unable to grow on glucose as the sole carbon source. The addition of ethanol or acetate in media containing glucose, but also raffinose or galactose, relieved this effect in batch cultivation, suggesting that the Crabtree effect is not the primary cause for the mutant's impaired growth on glucose. The addition of an energy source like formic acid restored glucose utilization, suggesting that a NADH/energy shortage in the Delta tpi1 mutant could be a cause of the impaired growth on glucose. The amount of glycerol production in the Delta tpi1 mutant could represent a good indicator of the fraction of carbon source channelled through glycolysis. Data obtained in continuous cultures on mixed substrates indicated that different contributions of glycolysis and gluconeogenesis, as well as of the HMP pathway, to glucose utilization by the Delta tpi1 mutant may occur in relation to the fraction of ethanol present in the media
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isolation nucleotide sequence and physiological relevance of the gene encoding triose phosphate Isomerase from kluyveromyces lactis
Applied and Environmental Microbiology, 1999Co-Authors: Concetta Compagno, Francesco Boschi, Danilo Porro, Agnese Daleffe, Bianca Maria RanziAbstract:Lack of triose phosphate Isomerase activity (TIM) is of special interest because this enzyme works at an important branch point of glycolytic flux. In this paper, we report the cloning and sequencing of the Kluyveromyces lactis gene encoding TIM. Unlike Saccharomyces cerevisiae ΔTPI1 mutants, the K. lactis mutant strain was found to be able to grow on glucose. Preliminary bioconversion experiments indicated that, like the S. cerevisiae TIM-deficient strain, the K. lactis TIM-deficient strain is able to produce glycerol with high yield.
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glycerol production in a triose phosphate Isomerase deficient mutant of saccharomyces cerevisiae
Biotechnology Progress, 1996Co-Authors: Concetta Compagno, Francesco Boschi, Bianca Maria RanziAbstract:Interesting challenges from metabolically engineered Saccharomyces cerevisiae cells arise from the opportunity to obtain yeast strains useful for the production of chemicals. In this paper, we show that engineered yeast cells deficient in the triose phosphate Isomerase activity are able to produce glycerol without the use of steering agents. High yields of conversion of glucose into glycerol (80-90% of the theoretical yield) and productivity (1.5 g L-1 h-1) have been obtained by a bioconversion process carried out in a poor and clean medium. We obtained indications that the growth phase at which the biomass was collected affect the process. The best results were obtained using cells collected at the end of exponential phase of growth. In perspective, the strategies and the information about the physiology of the cells described here could be useful for the developing of new biotechnological processes for glycerol production, outflanking the problems related to the use of high level of steering agents.
