The Experts below are selected from a list of 19344 Experts worldwide ranked by ideXlab platform
Paul A. Srere - One of the best experts on this subject based on the ideXlab platform.
-
interaction between Citrate Synthase and malate dehydrogenase substrate channeling of oxaloacetate
Journal of Biological Chemistry, 1998Co-Authors: Igor Morgunov, Paul A. SrereAbstract:Abstract The interactions between pig heart Citrate Synthase and mitochondrial malate dehydrogenase or cytosolic malate dehydrogenase were studied using the frontal analysis method of gel filtration and by precipitation in polyethylene glycol. This method showed that an interaction between Citrate Synthase and mitochondrial malate dehydrogenase occurred but no interaction between Citrate Synthase and cytosolic malate dehydrogenase. Channeling of oxaloacetate in the malate dehydrogenase and Citrate Synthase-coupled systems was tested using polyethylene glycol precipitates of Citrate Synthase and mitochondrial malate dehydrogenase, and Citrate Synthase and cytosolic malate dehydrogenase. The effectiveness of large amounts of aspartate aminotransferase and oxaloacetate decarboxylase, as competing enzymes for the intermediate oxaloacetate, was examined. Aspartate aminotransferase and oxaloacetate decarboxylase were less effective competitors for oxaloacetate when precipitated Citrate Synthase and mitochondrial malate dehydrogenase in polyethylene glycol was used at low ionic strength compared with free enzymes in the absence of polyethylene glycol or with a co-precipitate of Citrate Synthase and cytosolic malate dehydrogenase. Substrate channeling of oxaloacetate with Citrate Synthase-mitochondrial malate dehydrogenase precipitate was inefficient at high ionic strength. These effects could be explained through electrostatic interactions of mitochondrial but not cytosolic malate dehydrogenase with Citrate Synthase.
-
Azotobacter vinelandii Citrate Synthase.
Biochemistry, 1995Co-Authors: Magali Rault-leonardon, Mark A. L. Atkinson, Clive A Slaughter, Carolyn R Moomaw, Paul A. SrereAbstract:We have purified the Citrate Synthase from Azotobacter vinelandii and have determined that the size of the subunit is 48,000 Da and the structure of the holoenzyme is a hexamer. This contrasts with earlier estimates that indicate a 58,000 Da subunit and a tetrameric structure. In addition, the enzyme is allosteric with a Hill coefficient of 1.5 and is inhibited by NADH. The Hill coefficient is changed to about 1 by high ionic strength and AMP. The enzyme is thus similar to the Citrate Synthases of many other Gram-negative, facultative, anaerobic organisms. In addition, the amino acid sequence of about 100 residues has been determined and found to be highly similar to the sequence of Pseudomonas aeruginosa Citrate Synthase.
-
Conformational stability of pig Citrate Synthase and some active-site mutants
Biochemistry, 1991Co-Authors: Wang Zhi, Paul A. Srere, Claudia T. EvansAbstract:The conformational stabilities of native pig Citrate Synthase (PCS), a recombinant wild-type PCS, and six active-site mutant pig Citrate Synthases were studied in thermal denaturation experiments by circular dichroism and in urea denaturation experiments by using DTNB to measure the appearance of latent SH groups. His274 and Asp375 are conserved active-site residues in pig Citrate Synthase that bind to substrates and are implicated in the catalytic mechanism of the enzyme. By site-directed mutagenesis, His274 was replaced with Gly and Arg, while Asp375 was replaced with Gly, Asn, Glu, or Gln. These modifications were previously shown to result in 10(3)-10(4)-fold reductions in enzyme specific activities. The thermal unfolding of pig Citrate Synthase and the six mutants in the presence and absence of substrates showed large differences in the thermal stabilities of mutant proteins compared to the wild-type pig Citrate Synthase. The functions of His274 and Asp375 in ligand binding were measured by oxalacetate protection against urea denaturation. These data indicate that active-site mutations that decrease the specific activity of pig Citrate Synthase also cause an increase in the conformational stability of the protein. These results suggest that specific electrostatic interactions in the active site of Citrate Synthase are important in the catalytic mechanism in the chemical transformations as well as the conformational flexibility of the protein, both of which are important for the overall catalytic efficiency of the enzyme.
-
Studies on yeast peroxisomal Citrate Synthase.
Archives of biochemistry and biophysics, 1991Co-Authors: Gyula Kispal, Paul A. SrereAbstract:Peroxisomal (nonmitochondrial) Citrate Synthase (CS2) has been purified from a Saccharomyces cerevisiae strain in which the gene for the mitochondrial Citrate Synthase (CS1) had been disrupted and no CS1 protein is produced. The enzyme, CS2, the sequence of which had been previously determined from its DNA, behaved differently from CS1 in its purification, kinetics, stability, and binding to the inner surface of mitochondrial inner membranes.
David W. Hough - One of the best experts on this subject based on the ideXlab platform.
-
Rhodothermus marinus: a thermophilic bacterium producing dimeric and hexameric Citrate Synthase isoenzymes.
Extremophiles, 2002Co-Authors: Eva Nordberg Karlsson, Michael J. Danson, Olle Holst, Maher Abou-hachem, David W. HoughAbstract:Two separate Citrate Synthases from the extremely thermophilic bacterium Rhodothermus marinus have been identified and purified. One of the enzymes is a hexameric protein and is the first thermostable, hexameric Citrate Synthase to be isolated. The other is a dimeric enzyme, which is also thermostable but possesses both Citrate Synthase and 2-methyl Citrate Synthase activities. 2-Methyl Citrate Synthase uses propionyl-coenzyme A as one of its substrates and in Escherichia coli, for example, it has been implicated in the metabolism of propionate. However, no growth of R. marinus was observed using minimal medium with propionate as the sole carbon source, and both hexameric and dimeric enzymes were produced irrespective of whether propionate was included in the growth medium. The data are discussed with respect to the evolutionary relationships between the known hexameric and dimeric Citrate Synthases and 2-methyl Citrate Synthase.
-
structural adaptations of the cold active Citrate Synthase from an antarctic bacterium
Structure, 1998Co-Authors: Rupert J. M. Russell, Michael J. Danson, David W. Hough, Ursula Gerike, Garry L TaylorAbstract:Abstract Background: The structural basis of adaptation of enzymes to low temperature is poorly understood. Dimeric Citrate Synthase has been used as a model enzyme to study the structural basis of thermostability, the structure of the enzyme from organisms living in habitats at 55°C and 100°C having previously been determined. Here the study is extended to include a Citrate Synthase from an Antarctic bacterium, allowing us to explore the structural basis of cold activity and thermostability across the whole temperature range over which life is known to exist. Results: We report here the first crystal structure of a cold-active enzyme, Citrate Synthase, isolated from an Antarctic bacterium, at a resolution of 2.09 a. In comparison with the same enzyme from a hyperthermophilic host, the cold-active enzyme has a much more accessible active site, an unusual electrostatic potential distribution and an increased relative flexibility of the small domain compared to the large domain. Several other features of the cold-active enzyme were also identified: reduced subunit interface interactions with no intersubunit ion-pair networks; loops of increased length carrying more charge and fewer proline residues; an increase in solvent-exposed hydrophobic residues; and an increase in intramolecular ion pairs. Conclusions: Enzymes from organisms living at the temperature extremes of life need to avoid hot or cold denaturation yet maintain sufficient structural integrity to allow catalytic efficiency. For hyperthermophiles, thermal denaturation of the Citrate Synthase dimer appears to be resisted by complex networks of ion pairs at the dimer interface, a feature common to other hyperthermophilic proteins. For the cold-active Citrate Synthase, cold denaturation appears to be resisted by an increase in intramolecular ion pairs compared to the hyperthermophilic enzyme. Catalytic efficiency of the cold-active enzyme appears to be achieved by a more accessible active site and by an increase in the relative flexibility of the small domain compared to the large domain.
-
The Effect of Cysteine-43 Mutation on Thermostability and Kinetic Properties of Citrate Synthase from Thermoplasma acidophilum
Biochemical and Biophysical Research Communications, 1996Co-Authors: Semra Kocabıyık, İpek Erduran, Rupert J.m. Russel, Michael J. Danson, David W. HoughAbstract:Abstract In this study, we have substituted serine-43 by cysteine in the recombinant Citrate Synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutant Thermoplasma Citrate Synthase is very similar to wild-type Citrate Synthase in its substrate and co-factor specificities, pH profile and thermal stability. The mutation, however, has decreased the enzyme activity. The newly introduced reactive sulphydryl group could be easily modified by DTNB and labelled with 4-chloro-7-sulphobenzofuran, without loss of any activity.
-
Citrate Synthase from the hyperthermophilic Archaeon, Pyrococcus furiosus
Protein Engineering, 1995Co-Authors: Jacqueline M. Muir, Rupert J. M. Russell, David W. Hough, Michael J. DansonAbstract:The gene encoding the enzyme Citrate Synthase has been cloned and sequenced from the hyperthermophilic Archaeon Pyrococcus furiosus, and the derived amino acid sequence has been phylogenetically compared with Citrate Synthases from archaeal, bacterial and eukaryal organisms. The gene has been over-expressed in Escherichia coli to produce an active enzyme that has then been characterized with respect to its kinetic, oligomeric and hyperthermostable properties. A structurally-based sequence alignment was made to the Citrate Synthase from the thermophilic Archaeon Thermoplasma acidophilum, the crystal structure of which we have determined recently. From this alignment, a homology-modelled structure for the P.furiosus Citrate Synthase was generated and analysed.
-
the crystal structure of Citrate Synthase from the thermophilic archaeon thermoplasma acidophilum
Structure, 1994Co-Authors: Rupert J. M. Russell, Michael J. Danson, David W. Hough, G L TaylorAbstract:Abstract Background: The Archaea constitute a phylogenetically distinct, evolutionary domain and comprise organisms that live under environmental extremes of temperature, salinity and/or anaerobicity. Different members of the thermophilic Archaea tolerate temperatures in the range 55–110°C, and the comparison of the structures of their enzymes with the structurally homogolous enzymes of mesophilic organisms (optimum growth temperature range 15–45°C) may provide important information on the structural basis of protein thermostability. We have chosen Citrate Synthase, the first enzyme of the citric acid cycle, as a model enzyme for such studies. Results We have determined the crystal structure of Thermoplasma acidophilum Citrate Synthase to 2.5 A and have compared it with the Citrate Synthase from pig heart, with which it shares a high degree of structural homology, but little sequence identity (20%). Conclusion The three-dimensional structural comparison of thermophilic and mesophilic Citrate Synthases has permitted catalytic and substrate-binding residues to be tentatively assigned in the archaeal, thermophilic enzyme, and has identified structural features that may be responsible for its thermostability.
Michael J. Danson - One of the best experts on this subject based on the ideXlab platform.
-
Rhodothermus marinus: a thermophilic bacterium producing dimeric and hexameric Citrate Synthase isoenzymes.
Extremophiles, 2002Co-Authors: Eva Nordberg Karlsson, Michael J. Danson, Olle Holst, Maher Abou-hachem, David W. HoughAbstract:Two separate Citrate Synthases from the extremely thermophilic bacterium Rhodothermus marinus have been identified and purified. One of the enzymes is a hexameric protein and is the first thermostable, hexameric Citrate Synthase to be isolated. The other is a dimeric enzyme, which is also thermostable but possesses both Citrate Synthase and 2-methyl Citrate Synthase activities. 2-Methyl Citrate Synthase uses propionyl-coenzyme A as one of its substrates and in Escherichia coli, for example, it has been implicated in the metabolism of propionate. However, no growth of R. marinus was observed using minimal medium with propionate as the sole carbon source, and both hexameric and dimeric enzymes were produced irrespective of whether propionate was included in the growth medium. The data are discussed with respect to the evolutionary relationships between the known hexameric and dimeric Citrate Synthases and 2-methyl Citrate Synthase.
-
structural adaptations of the cold active Citrate Synthase from an antarctic bacterium
Structure, 1998Co-Authors: Rupert J. M. Russell, Michael J. Danson, David W. Hough, Ursula Gerike, Garry L TaylorAbstract:Abstract Background: The structural basis of adaptation of enzymes to low temperature is poorly understood. Dimeric Citrate Synthase has been used as a model enzyme to study the structural basis of thermostability, the structure of the enzyme from organisms living in habitats at 55°C and 100°C having previously been determined. Here the study is extended to include a Citrate Synthase from an Antarctic bacterium, allowing us to explore the structural basis of cold activity and thermostability across the whole temperature range over which life is known to exist. Results: We report here the first crystal structure of a cold-active enzyme, Citrate Synthase, isolated from an Antarctic bacterium, at a resolution of 2.09 a. In comparison with the same enzyme from a hyperthermophilic host, the cold-active enzyme has a much more accessible active site, an unusual electrostatic potential distribution and an increased relative flexibility of the small domain compared to the large domain. Several other features of the cold-active enzyme were also identified: reduced subunit interface interactions with no intersubunit ion-pair networks; loops of increased length carrying more charge and fewer proline residues; an increase in solvent-exposed hydrophobic residues; and an increase in intramolecular ion pairs. Conclusions: Enzymes from organisms living at the temperature extremes of life need to avoid hot or cold denaturation yet maintain sufficient structural integrity to allow catalytic efficiency. For hyperthermophiles, thermal denaturation of the Citrate Synthase dimer appears to be resisted by complex networks of ion pairs at the dimer interface, a feature common to other hyperthermophilic proteins. For the cold-active Citrate Synthase, cold denaturation appears to be resisted by an increase in intramolecular ion pairs compared to the hyperthermophilic enzyme. Catalytic efficiency of the cold-active enzyme appears to be achieved by a more accessible active site and by an increase in the relative flexibility of the small domain compared to the large domain.
-
Citrate Synthase and 2-methylCitrate Synthase: structural, functional and evolutionary relationships.
Microbiology (Reading England), 1998Co-Authors: U Gerike, D W Hough, N J Russell, M L Dyall-smith, Michael J. DansonAbstract:Following the complete sequencing of the Escherichia coli genome, it has been shown that the proposed second Citrate Synthase of this organism, recently described by the authors, is in fact a 2-methylCitrate Synthase that possesses Citrate Synthase activity as a minor component. Whereas the hexameric Citrate Synthase is constitutively produced, the 2-methylCitrate Synthase is induced during growth on propionate, and the catabolism of propionate to succinate and pyruvate via 2-methylCitrate is proposed. The Citrate Synthases of the psychrotolerant eubacterium DS2-3R, and of the thermophilic archaea Thermoplasma acidophilum and Pyrococcus furiosus, are approximately 40% identical in sequence to the Escherichia coli 2-methylCitrate Synthase and also possess 2-methylCitrate Synthase activity. The data are discussed with respect to the structure, function and evolution of Citrate Synthase and 2-methylCitrate Synthase.
-
The Effect of Cysteine-43 Mutation on Thermostability and Kinetic Properties of Citrate Synthase from Thermoplasma acidophilum
Biochemical and Biophysical Research Communications, 1996Co-Authors: Semra Kocabıyık, İpek Erduran, Rupert J.m. Russel, Michael J. Danson, David W. HoughAbstract:Abstract In this study, we have substituted serine-43 by cysteine in the recombinant Citrate Synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutant Thermoplasma Citrate Synthase is very similar to wild-type Citrate Synthase in its substrate and co-factor specificities, pH profile and thermal stability. The mutation, however, has decreased the enzyme activity. The newly introduced reactive sulphydryl group could be easily modified by DTNB and labelled with 4-chloro-7-sulphobenzofuran, without loss of any activity.
-
Citrate Synthase from the hyperthermophilic Archaeon, Pyrococcus furiosus
Protein Engineering, 1995Co-Authors: Jacqueline M. Muir, Rupert J. M. Russell, David W. Hough, Michael J. DansonAbstract:The gene encoding the enzyme Citrate Synthase has been cloned and sequenced from the hyperthermophilic Archaeon Pyrococcus furiosus, and the derived amino acid sequence has been phylogenetically compared with Citrate Synthases from archaeal, bacterial and eukaryal organisms. The gene has been over-expressed in Escherichia coli to produce an active enzyme that has then been characterized with respect to its kinetic, oligomeric and hyperthermostable properties. A structurally-based sequence alignment was made to the Citrate Synthase from the thermophilic Archaeon Thermoplasma acidophilum, the crystal structure of which we have determined recently. From this alignment, a homology-modelled structure for the P.furiosus Citrate Synthase was generated and analysed.
Claudia T. Evans - One of the best experts on this subject based on the ideXlab platform.
-
Role of Citrate Synthase in Aldosterone-Mediated Sodium Reabsorption
Hypertension (Dallas Tex. : 1979), 2000Co-Authors: Michael E. Ullian, Claudia T. Evans, Christopher J. Robinson, Joel Z. Melnick, Wayne R. FitzgibbonAbstract:Abstract —Aldosterone and other mineralocorticoids increase Citrate Synthase activity in the kidney and enhance renal sodium reabsorption, but it is unclear whether the increased Citrate Synthase activity is involved in renal sodium transport. We used the Wistar-Furth rat, an inbred strain found to be deficient in renal Citrate Synthase activity, as an experimental model to investigate this issue. We confirmed that renal Citrate Synthase activity from adrenalectomized Wistar-Furth rats was decreased compared with that from control Wistar rats (by 28%). Similarly, urinary Citrate excretion was 23% lower in Wistar-Furth rats. Subnormal Citrate formation in Wistar-Furth rats could not be accounted for by differences in systemic pH or circulating potassium levels. Because renal Citrate Synthase activity was reduced in Wistar-Furth rats, we hypothesized that renal sodium excretory responses to mineralocorticoids would be reduced as well. Four-hour sodium excretion after intraperitoneal injection of 5 μg of aldosterone was reduced by 56% in adrenalectomized Wistar rats and by 52% in adrenalectomized Wistar-Furth rats (both P
-
Metabolic engineering of a non-allosteric Citrate Synthase in an Escherichia coli Citrate Synthase mutant.
Journal of molecular recognition : JMR, 1995Co-Authors: Claudia T. EvansAbstract:This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high-resolution 13C NMR. The oxidation of [1,2,3-13C]propionate to glutamate via the TCA cycle was measured in wild-type (WT) and a Citrate Synthase mutant (CS-) strain of Escherichia coli transformed with allosteric E. coli Citrate Synthase (ECCS) or non-allosteric pig Citrate Synthase (PCS). The 13C fractional enrichment in glutamate C-2, C-3, and C-4 in ECCS and PCS were similar; although quantitative differences in total Citrate Synthase activity and total C-4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10-fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C-4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure (TCACALC) and yielded an acetate/succinyl-CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the 13C spectra of cells presented with [3-13C]propionate or [2-13C]propionate. The results are consistent with the presence of an allosteric Citrate Synthase in ECCS and a non-allosteric Citrate Synthase in PCS. The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels.
-
Conformational stability of pig Citrate Synthase and some active-site mutants
Biochemistry, 1991Co-Authors: Wang Zhi, Paul A. Srere, Claudia T. EvansAbstract:The conformational stabilities of native pig Citrate Synthase (PCS), a recombinant wild-type PCS, and six active-site mutant pig Citrate Synthases were studied in thermal denaturation experiments by circular dichroism and in urea denaturation experiments by using DTNB to measure the appearance of latent SH groups. His274 and Asp375 are conserved active-site residues in pig Citrate Synthase that bind to substrates and are implicated in the catalytic mechanism of the enzyme. By site-directed mutagenesis, His274 was replaced with Gly and Arg, while Asp375 was replaced with Gly, Asn, Glu, or Gln. These modifications were previously shown to result in 10(3)-10(4)-fold reductions in enzyme specific activities. The thermal unfolding of pig Citrate Synthase and the six mutants in the presence and absence of substrates showed large differences in the thermal stabilities of mutant proteins compared to the wild-type pig Citrate Synthase. The functions of His274 and Asp375 in ligand binding were measured by oxalacetate protection against urea denaturation. These data indicate that active-site mutations that decrease the specific activity of pig Citrate Synthase also cause an increase in the conformational stability of the protein. These results suggest that specific electrostatic interactions in the active site of Citrate Synthase are important in the catalytic mechanism in the chemical transformations as well as the conformational flexibility of the protein, both of which are important for the overall catalytic efficiency of the enzyme.
Marco Aurelio Pardo - One of the best experts on this subject based on the ideXlab platform.
-
Rhizobium tropici chromosomal Citrate Synthase gene
Applied and environmental microbiology, 1995Co-Authors: Ismael Hernández-lucas, Marco Aurelio Pardo, Lorenzo Segovia, J Miranda, Esperanza Martínez-romeroAbstract:Two genes encoding Citrate Synthase, a key enzyme in the Krebs cycle, have been found in Rhizobium tropici. One of them is in the bacterial chromosome, while the other is in the symbiotic plasmid. We sequenced the chromosomal gene and found that it is very similar to the previously reported plasmidic gene sequence in its structural region but not in its regulatory region. The chromosomal gene is able to complement an Escherichia coli Citrate Synthase mutant. In R. tropici, a mutant in the chromosomal Citrate Synthase gene has a diminished Citrate Synthase activity (in free-living bacteria), a diminished nodulation capacity, and forms nitrogen-fixing nodules. In contrast, the Citrate Synthase double mutant forms ineffective nodules devoid of bacteroids and forms less nodules than the single chromosomal mutant. It is inferred that both genes are functional and required during the nodulation process in R. tropici.
-
Nodulating ability of Rhizobium tropici is conditioned by a plasmid-encoded Citrate Synthase
Molecular microbiology, 1994Co-Authors: Marco Aurelio Pardo, J Miranda, Jaime Lagunez, Esperanza MartinezAbstract:Summary Rhizobium species elicit the formation of nitrogen-fixing root nodules through a complex interaction between bacteria and plants. Various bacterial genes involved in the nodulation and nitrogen-fixation processes have been described and most have been localized on the symbiotic plasmids (pSym). We have found a gene encoding Citrate Synthase on the pSym plasmid of Rhizobium tropici, a species that forms nitrogen-fixing nodules on the roots of beans (PhasBoius vuigaris) and trees (Leucaena spp.). Citrate Synthase is a key metabolic enzyme that incorporates carbon into the tricarboxylic acid cycle by catalysing the condensation of acetyl-CoA and oxalo-acetic acid to form Citrate. R. tropici pcsA (the plasmid Citrate Synthase gene) is closely related to the corresponding genes of Proteobacteria. pcsA inactivation by a Tn5-mob insertion causes the bacteria to form fewer nodules (30–50% of the original strain) and to have a decreased Citrate Synthase activity in minimal medium with sucrose. A clone carrying the pcsA gene complemented ail the phenotypic alterations of the pcsA mutant, and conferred Rhizobium iegumino-sarum bv. phaseoli (which naturally lacks a plasmid Citrate Synthase gene) a higher nodulation and growth capacity in correlation with a higher Citrate Synthase activity. We have also found that pcsA gene expression is sensitive to iron availability, suggesting a possible role of pcsA in iron uptake.