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Frank M. Raushel - One of the best experts on this subject based on the ideXlab platform.

  • Carbamate Transport in Carbamoyl Phosphate Synthetase: A Theoretical and Experimental Investigation
    Journal of the American Chemical Society, 2010
    Co-Authors: Liliya Lund, Yubo Fan, Qiang Shao, Yi Qin Gao, Frank M. Raushel
    Abstract:

    The transport of carbamate through the large subunit of Carbamoyl Phosphate Synthetase (CPS) from Escherichia coli was investigated by molecular dynamics and site-directed mutagenesis. Carbamate, the product of the reaction involving ATP, bicarbonate, and ammonia, must be delivered from the site of formation to the site of utilization by traveling nearly 40 A within the enzyme. Potentials of mean force (PMF) calculations along the entire tunnel for the translocation of carbamate indicate that the tunnel is composed of three continuous water pockets and two narrow connecting parts, near Ala-23 and Gly-575. The two narrow parts render two free energy barriers of 6.7 and 8.4 kcal/mol, respectively. Three water pockets were filled with about 21, 9, and 9 waters, respectively, and the corresponding relative free energies of carbamate residing in these free energy minima are 5.8, 0, and 1.6 kcal/mol, respectively. The release of Phosphate into solution at the site for the formation of carbamate allows the side ...

  • Access to the carbamate tunnel of Carbamoyl Phosphate Synthetase
    Archives of biochemistry and biophysics, 2004
    Co-Authors: Jungwook Kim, Frank M. Raushel
    Abstract:

    The X-ray crystal structure of Carbamoyl Phosphate Synthetase (CPS) from Escherichia coli revealed the existence of a molecular tunnel that has been proposed to facilitate the translocation of reaction intermediates between remotely located active sites. Five highly conserved glutamate residues, including Glu-25, Glu-383, Glu-577, Glu-604, and Glu-916, are close together in two clusters in the interior wall of the molecular tunnel that enables the intermediate carbamate to migrate from the site of synthesis to the site of utilization. Two arginines, Arg-306 and Arg-848, are located at either end of the carbamate tunnel and participate in the binding of ATP at each of the two active sites within the large subunit of CPS. The mutation of Glu-25 or Glu-577 results in a diminution in the overall rate of Carbamoyl Phosphate formation. Similar effects are observed upon mutation of Arg-306 and Arg-848 to alanine residues. The conserved glutamate and arginine residues may function in concert with one another to control entry of carbamate into the tunnel prior to phosphorylation to Carbamoyl Phosphate. The electrostatic environment of tunnel interior may help to stabilize the tunnel architecture and prevent decomposition of carbamate through protonation.

  • Long-range allosteric transitions in Carbamoyl Phosphate Synthetase.
    Protein science : a publication of the Protein Society, 2004
    Co-Authors: James B. Thoden, Xinyi Huang, Jungwook Kim, Frank M. Raushel, Hazel M. Holden
    Abstract:

    Carbamoyl Phosphate Synthetase plays a key role in both pyrimidine and arginine biosynthesis by catalyzing the production of Carbamoyl Phosphate from one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli consists of two polypeptide chains referred to as the small and large subunits, which contain a total of three separate active sites that are connected by an intramolecular tunnel. The small subunit harbors one of these active sites and is responsible for the hydrolysis of glutamine to glutamate and ammonia. The large subunit binds the two required molecules of MgATP and is involved in assembling the final product. Compounds such as L-ornithine, UMP, and IMP allosterically regulate the enzyme. Here, we report the three-dimensional structure of a site-directed mutant protein of Carbamoyl Phosphate Synthetase from E. coli, where Cys 248 in the small subunit was changed to an aspartate. This residue was targeted for a structural investigation because previous studies demonstrated that the partial glutaminase activity of the C248D mutant protein was increased 40-fold relative to the wild-type enzyme, whereas the formation of Carbamoyl Phosphate using glutamine as a nitrogen source was completely abolished. Remarkably, although Cys 248 in the small subunit is located at ∼100 A from the allosteric binding pocket in the large subunit, the electron density map clearly revealed the presence of UMP, although this ligand was never included in the purification or crystallization schemes. The manner in which UMP binds to Carbamoyl Phosphate Synthetase is described.

  • Structural defects within the carbamate tunnel of Carbamoyl Phosphate Synthetase.
    Biochemistry, 2002
    Co-Authors: Jungwook Kim, Xinyi Huang, Stanley C. Howell, Frank M. Raushel
    Abstract:

    The X-ray crystal structure of Carbamoyl Phosphate Synthetase (CPS) from Escherichia coli has unveiled the existence of two molecular tunnels within the heterodimeric enzyme. These two interdomain tunnels connect the three distinct active sites within this remarkably complex protein and apparently function as conduits for the transport of unstable reaction intermediates between successive active sites. The operational significance of the ammonia tunnel for the migration of NH3 is supported experimentally by isotope competition and protein modification. The passage of carbamate through the carbamate tunnel has now been assessed by the insertion of site-directed structural blockages within this tunnel. Gln-22, Ala-23, and Gly-575 from the large subunit of CPS were substituted by mutagenesis with bulkier amino acids in an attempt to obstruct and/or hinder the passage of the unstable intermediate through the carbamate tunnel. The structurally modified proteins G575L, A23L/G575S, and A23L/G575L exhibited a sub...

  • Carbamoyl-Phosphate Synthetase. Creation of an escape route for ammonia.
    The Journal of biological chemistry, 2002
    Co-Authors: James B. Thoden, Xinyi Huang, Frank M. Raushel, Hazel M. Holden
    Abstract:

    Abstract Carbamoyl-Phosphate Synthetase catalyzes the production of Carbamoyl Phosphate through a reaction mechanism requiring one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli is composed of two polypeptide chains. The smaller of these belongs to the Class I amidotransferase superfamily and contains all of the necessary amino acid side chains required for the hydrolysis of glutamine to glutamate and ammonia. Two homologous domains from the larger subunit adopt conformations that are characteristic for members of the ATP-grasp superfamily. Each of these ATP-grasp domains contains an active site responsible for binding one molecule of MgATP. High resolution x-ray crystallographic analyses have shown that, remarkably, the three active sites in the E. coli enzyme are connected by a molecular tunnel of ∼100 A in total length. Here we describe the high resolution x-ray crystallographic structure of the G359F (small subunit) mutant protein of Carbamoyl Phosphate Synthetase. This residue was initially targeted for study because it resides within the interior wall of the molecular tunnel leading from the active site of the small subunit to the first active site of the large subunit. It was anticipated that a mutation to the larger residue would “clog” the ammonia tunnel and impede the delivery of ammonia from its site of production to the site of utilization. In fact, the G359F substitution resulted in a complete change in the conformation of the loop delineated by Glu-355 to Ala-364, thereby providing an “escape” route for the ammonia intermediate directly to the bulk solvent. The substitution also effected the disposition of several key catalytic amino acid side chains in the small subunit active site.

Guy Hervé - One of the best experts on this subject based on the ideXlab platform.

Cristina Purcarea - One of the best experts on this subject based on the ideXlab platform.

  • Direct demonstration of Carbamoyl Phosphate formation on the C-terminal domain of Carbamoyl Phosphate Synthetase.
    Protein science : a publication of the Protein Society, 2004
    Co-Authors: Michael Kothe, Cristina Purcarea, Hedeel I. Guy, David R. Evans, S G Powers-lee
    Abstract:

    Carbamoyl Phosphate Synthetase synchronizes the utilization of two ATP molecules at duplicated ATP-grasp folds to catalyze Carbamoyl Phosphate formation. To define the dedicated functional role played by each of the two ATP sites, we have carried out pulse/labeling studies using the Synthetases from Aquifex aeolicus and Methanococcus jannaschii, hyperthermophilic organisms that encode the two ATP-grasp folds on separate subunits. These studies allowed us to differentially label each active site with [γ-32P]ATP and determine the fate of the labeled γ-Phosphate in the Synthetase reaction. Our results provide the first direct demonstration that enzyme-catalyzed transfer of Phosphate from ATP to carbamate occurs on the more C-terminal of the two ATP-grasp folds. These findings rule out one mechanism proposed for Carbamoyl Phosphate Synthetase, where one ATP acts as a molecular switch, and provide additional support for a sequential reaction mechanism where the γ-Phosphate groups of both ATP molecules are transferred to reactants. CP synthesis by subunit C in our single turnover pulse/chase assays did not require subunit N, but subunit N was required for detectable CP synthesis in the traditional continuous assay. These findings suggest that cross-talk between domain N and C is required for product release from subunit C.

  • a novel Carbamoyl Phosphate Synthetase from aquifex aeolicus
    Journal of Biological Chemistry, 2001
    Co-Authors: Anupama Ahuja, Cristina Purcarea, Hedeel I. Guy, David R. Evans
    Abstract:

    Abstract Aquifex aeolicus, an extreme hyperthermophile, has neither a full-length Carbamoyl-Phosphate Synthetase (CPSase) resembling the enzyme found in all mesophilic organisms nor a carbamate kinase-like CPSase such as those present in several hyperthermophilic archaea. However, the genome has open reading frames encoding putative proteins that are homologous to the major CPSase domains. The glutaminase, CPS.A, and CPS.B homologs from A. aeolicus were cloned, overexpressed inEscherichia coli, and purified to homogeneity. The isolated proteins could catalyze several partial reactions but not the overall synthesis of Carbamoyl Phosphate. However, a stable 124-kDa complex could be reconstituted from stoichiometric amounts of CPS.A and CPS.B proteins that synthesized Carbamoyl Phosphate from ATP, bicarbonate, and ammonia. The inclusion of the glutaminase subunit resulted in the formation of a 171-kDa complex that could utilize glutamine as the nitrogen-donating substrate, although the catalytic efficiency was significantly compromised. Molecular modeling, using E. coli CPSase as a template, showed that the enzyme has a similar structural organization and interdomain interfaces and that all of the residues known to be essential for function are conserved and properly positioned. A steady state kinetic study at 78 °C indicated that although the substrate affinity was similar for bicarbonate, ammonia, and glutamine, the K m for ATP was appreciably higher than that of any known CPSase. The A. aeolicuscomplex, with a split gene encoding the major Synthetase domains and relatively inefficient coupling of amidotransferase and Synthetase functions, may be more closely related to the ancestral precursor of contemporary mesophilic CPSases.

  • Purification and Characterization of Carbamoyl‐Phosphate Synthetase from the Deep‐Sea Hyperthermophilic Archaebacterium Pyrococcus abyssi
    FEBS Journal, 1996
    Co-Authors: Cristina Purcarea, Daniel Prieur, Valérie Simon, Guy Hervé
    Abstract:

    Carbamoyl-Phosphate Synthetase was purified from the deep-sea hyperthermophilic archaebacterium Pyrococcus abyssi. This enzyme appears to be monomeric and uses ammonium salts as nitrogen donor. Its activity is inhibited by some nucleotides that compete with ATP. In contrast with the Carbamoyl-Phosphate Synthetases investigated so far, this enzyme is very resistant to high temperature. Its low molecular mass (46.6 kDa) and its catalytic properties suggest that the gene coding for this enzyme is a previously postulated ancestor whose duplication gave the genes coding for Carbamoyl-Phosphate Synthetases and carbamate kinases.

  • purification and characterization of Carbamoyl Phosphate Synthetase from the deep sea hyperthermophilic archaebacterium pyrococcus abyssi
    FEBS Journal, 1996
    Co-Authors: Cristina Purcarea, Daniel Prieur, Valérie Simon, Guy Hervé
    Abstract:

    Carbamoyl-Phosphate Synthetase was purified from the deep-sea hyperthermophilic archaebacterium Pyrococcus abyssi. This enzyme appears to be monomeric and uses ammonium salts as nitrogen donor. Its activity is inhibited by some nucleotides that compete with ATP. In contrast with the Carbamoyl-Phosphate Synthetases investigated so far, this enzyme is very resistant to high temperature. Its low molecular mass (46.6 kDa) and its catalytic properties suggest that the gene coding for this enzyme is a previously postulated ancestor whose duplication gave the genes coding for Carbamoyl-Phosphate Synthetases and carbamate kinases.

Eduardo Villamor - One of the best experts on this subject based on the ideXlab platform.

  • The T1405N Carbamoyl Phosphate Synthetase Polymorphism Does Not Affect Plasma Arginine Concentrations in Preterm Infants
    PloS one, 2010
    Co-Authors: Rob M. Moonen, I Reyes, Giacomo Cavallaro, Gema E. González-luis, Jaap A. Bakker, Eduardo Villamor
    Abstract:

    Background A C-to-A nucleotide transversion (T1405N) in the gene that encodes Carbamoyl-Phosphate Synthetase 1 (CPS1) has been associated with changes in plasma concentrations of L-arginine in term and near term infants but not in adults. In preterm infants homozygosity for the CPS1 Thr1405 variant (CC genotype) was associated with an increased risk of having necrotizing enterocolitis (NEC). Plasma L-arginine concentrations are decreased in preterm infants with NEC.

  • 383 The T1405N Carbamoyl Phosphate Synthetase Polymorphism Does Not Affect Plasma Arginine Concentrations in Preterm Infants
    Pediatric Research, 2010
    Co-Authors: Rob M. Moonen, I Reyes, Giacomo Cavallaro, Gema E. González-luis, Jaap A. Bakker, Eduardo Villamor
    Abstract:

    383 The T1405N Carbamoyl Phosphate Synthetase Polymorphism Does Not Affect Plasma Arginine Concentrations in Preterm Infants

  • Carbamoyl Phosphate Synthetase polymorphisms as a risk factor for necrotizing enterocolitis
    Pediatric Research, 2007
    Co-Authors: Rob M. Moonen, Aimee D C Paulussen, N Y Souren, Alfons G H Kessels, Estela M Rubiogozalbo, Eduardo Villamor
    Abstract:

    A C-to-A nucleotide transversion (T1405N) in the gene that encodes Carbamoyl-Phosphate Synthetase 1 (CPS1) has been correlated with low plasma concentrations of l-arginine in neonates. As plasma l-arginine concentrations are decreased in premature infants with necrotizing enterocolitis (NEC), we hypothesized that the CPS1 T1405N polymorphism would correlate with the presence of NEC. We analyzed the CPS1 genotypes for the T1405N polymorphism in 17 preterm infants (≤30 wk and <1500 g) with established NEC, 34 preterm infants without NEC, and 25 healthy term infants. Distribution of genotypes did not differ between the NEC population (CC:AC:AA = 70.6%:23.5%:5.9%) and the preterm control group (CC:AC:AA = 41.2%:35.3%:23.5%; p = 0.110) or the term group (CC:AC:AA = 44%:48%:8%; p = 0.228). The C allele frequency was 82.4% in NEC and 58.8% in preterm control infants (p = 0.018) and analysis for linear trend demonstrated that incidence of NEC increased with the number of C alleles (p = 0.037). The CC genotype was associated with an increased risk of NEC in the preterm infants [odds ratio (OR) = 3.43, 95% confidence interval (CI): 1.01–11.49, p = 0.048), when compared with the grouped together AA/AC genotypes. These data suggest that the CPS1 T1405N polymorphism may be associated with the risk of NEC in preterm infants.

Hazel M. Holden - One of the best experts on this subject based on the ideXlab platform.

  • Long-range allosteric transitions in Carbamoyl Phosphate Synthetase.
    Protein science : a publication of the Protein Society, 2004
    Co-Authors: James B. Thoden, Xinyi Huang, Jungwook Kim, Frank M. Raushel, Hazel M. Holden
    Abstract:

    Carbamoyl Phosphate Synthetase plays a key role in both pyrimidine and arginine biosynthesis by catalyzing the production of Carbamoyl Phosphate from one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli consists of two polypeptide chains referred to as the small and large subunits, which contain a total of three separate active sites that are connected by an intramolecular tunnel. The small subunit harbors one of these active sites and is responsible for the hydrolysis of glutamine to glutamate and ammonia. The large subunit binds the two required molecules of MgATP and is involved in assembling the final product. Compounds such as L-ornithine, UMP, and IMP allosterically regulate the enzyme. Here, we report the three-dimensional structure of a site-directed mutant protein of Carbamoyl Phosphate Synthetase from E. coli, where Cys 248 in the small subunit was changed to an aspartate. This residue was targeted for a structural investigation because previous studies demonstrated that the partial glutaminase activity of the C248D mutant protein was increased 40-fold relative to the wild-type enzyme, whereas the formation of Carbamoyl Phosphate using glutamine as a nitrogen source was completely abolished. Remarkably, although Cys 248 in the small subunit is located at ∼100 A from the allosteric binding pocket in the large subunit, the electron density map clearly revealed the presence of UMP, although this ligand was never included in the purification or crystallization schemes. The manner in which UMP binds to Carbamoyl Phosphate Synthetase is described.

  • Carbamoyl-Phosphate Synthetase. Creation of an escape route for ammonia.
    The Journal of biological chemistry, 2002
    Co-Authors: James B. Thoden, Xinyi Huang, Frank M. Raushel, Hazel M. Holden
    Abstract:

    Abstract Carbamoyl-Phosphate Synthetase catalyzes the production of Carbamoyl Phosphate through a reaction mechanism requiring one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli is composed of two polypeptide chains. The smaller of these belongs to the Class I amidotransferase superfamily and contains all of the necessary amino acid side chains required for the hydrolysis of glutamine to glutamate and ammonia. Two homologous domains from the larger subunit adopt conformations that are characteristic for members of the ATP-grasp superfamily. Each of these ATP-grasp domains contains an active site responsible for binding one molecule of MgATP. High resolution x-ray crystallographic analyses have shown that, remarkably, the three active sites in the E. coli enzyme are connected by a molecular tunnel of ∼100 A in total length. Here we describe the high resolution x-ray crystallographic structure of the G359F (small subunit) mutant protein of Carbamoyl Phosphate Synthetase. This residue was initially targeted for study because it resides within the interior wall of the molecular tunnel leading from the active site of the small subunit to the first active site of the large subunit. It was anticipated that a mutation to the larger residue would “clog” the ammonia tunnel and impede the delivery of ammonia from its site of production to the site of utilization. In fact, the G359F substitution resulted in a complete change in the conformation of the loop delineated by Glu-355 to Ala-364, thereby providing an “escape” route for the ammonia intermediate directly to the bulk solvent. The substitution also effected the disposition of several key catalytic amino acid side chains in the small subunit active site.

  • The structure of Carbamoyl Phosphate Synthetase determined to 2.1 Å resolution
    Acta Crystallographica Section D Biological Crystallography, 1999
    Co-Authors: James B. Thoden, Frank M. Raushel, Matthew M. Benning, Ivan Rayment, Hazel M. Holden
    Abstract:

    Carbamoyl Phosphate Synthetase catalyzes the formation of Carbamoyl Phosphate from one molecule of bicarbonate, two molecules of Mg2+ATP and one molecule of glutamine or ammonia depending upon the particular form of the enzyme under investigation. As isolated from Escherichia coli, the enzyme is an \alpha,β-heterodimer consisting of a small subunit that hydrolyzes glutamine and a large subunit that catalyzes the two required phosphorylation events. Here the three-dimensional structure of Carbamoyl Phosphate Synthetase from E. coli refined to 2.1 A resolution with an R factor of 17.9% is described. The small subunit is distinctly bilobal with a catalytic triad (Cys269, His353 and Glu355) situated between the two structural domains. As observed in those enzymes belonging to the \alpha/\beta-hydrolase family, the active-site nucleophile, Cys269, is perched at the top of a tight turn. The large subunit consists of four structural units: the carboxyPhosphate synthetic component, the oligomerization domain, the Carbamoyl Phosphate synthetic component and the allosteric domain. Both the carboxyPhosphate and Carbamoyl Phosphate synthetic components bind Mn2+ADP. In the carboxyPhosphate synthetic component, the two observed Mn2+ ions are both octahedrally coordinated by oxygen-containing ligands and are bridged by the carboxylate side chain of Glu299. Glu215 plays a key allosteric role by coordinating to the physiologically important potassium ion and hydrogen bonding to the ribose hydroxyl groups of ADP. In the Carbamoyl Phosphate synthetic component, the single observed Mn2+ ion is also octahedrally coordinated by oxygen-containing ligands and Glu761 plays a similar role to that of Glu215. The carboxy­Phosphate and Carbamoyl Phosphate synthetic components, while topologically equivalent, are structurally different, as would be expected in light of their separate biochemical functions.

  • Carbamoyl Phosphate Synthetase: caught in the act of glutamine hydrolysis.
    Biochemistry, 1998
    Co-Authors: James B. Thoden, Frank M. Raushel, Sophie Gaillard Miran, James C. Phillips, Andrew Howard, Hazel M. Holden
    Abstract:

    Carbamoyl Phosphate Synthetase from Escherichia coli catalyzes the production of Carbamoyl Phosphate from two molecules of Mg 2+ ATP, one molecule of bicarbonate, and one molecule of glutamine. The enzyme consists of two polypeptide chains referred to as the large and small subunits. While the large subunit provides the active sites responsible for the binding of nucleotides and other effector ligands, the small subunit contains those amino acid residues that catalyze the hydrolysis of glutamine to glutamate and ammonia. From both amino acid sequence analyses and structural studies it is now known that the small subunit belongs to the class I amidotransferase family of enzymes. Numerous biochemical studies have suggested that the reaction mechanism of the small subunit proceeds through the formation of the glutamyl thioester intermediate and that both Cys 269 and His 353 are critical for catalysis. Here we describe the X-ray crystallographic structure of Carbamoyl Phosphate Synthetase from E. coli in which His 353 has been replaced with an asparagine residue. Crystals employed in the investigation were grown in the presence of glutamine, and the model has been refined to a crystallographic R-factor of 19.1% for all measured X-ray data from 30 to 1.8 A resolution. The active site of the small subunit clearly contains a covalently bound thioester intermediate at Cys 269, and indeed, this investigation provides the first direct structural observation of an enzyme intermediate in the amidotransferase family.

  • Carbamoyl Phosphate Synthetase: a tunnel runs through it.
    Current opinion in structural biology, 1998
    Co-Authors: Hazel M. Holden, James B. Thoden, Frank M. Raushel
    Abstract:

    The direct transfer of metabolites from one protein to another in a biochemical pathway or between one active site and another within a single enzyme has been described as substrate channeling. The first structural visualization of such a phenomenon was provided by the X-ray crystallographic analysis of tryptophan synthase, in which a tunnel of approximately 25 A in length was observed. The recently determined three-dimensional structure of Carbamoyl Phosphate Synthetase sets a new long distance record in that the three active sites are separated by nearly 100 A.