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Frank M. Raushel - One of the best experts on this subject based on the ideXlab platform.
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Biological Crystallography The structure of Carbamoyl Phosphate synthetase determined to 2.1 A Ê resolution
2020Co-Authors: James B. Thoden, Frank M. Raushel, Matthew M. Benning, Ivan Rayment, Hazel M. HoldenAbstract:Carbamoyl Phosphate synthetase catalyzes the formation of Carbamoyl Phosphate from one molecule of bicarbonate, two molecules of Mg 2+ 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 ,-heterodimer consisting of a small subunit that hydrolyzes glutamine and a large subunit that catalyzes the two required phosphorylation events. Here the threedimensional structure of Carbamoyl Phosphate synthetase from E. coli re®ned to 2.1 A Ê 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 a-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 Mn 2+ ADP. In the carboxyPhosphate synthetic component, the two observed Mn 2+ 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 Mn 2+ 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
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Dissection of the Conduit for Allosteric Control of Carbamoyl Phosphate Synthetase by Ornithine
2020Co-Authors: Olivier A Pierrat, Farah Javid-majd, Frank M. RaushelAbstract:Ornithine is an allosteric activator of Carbamoyl Phosphate synthetase (CPS) from Escherichia coli. Nine amino acids in the vicinity of the binding sites for ornithine and potassium were mutated to alanine, glutamine, or lysine. The residues E783, T1042, and T1043 were found to be primarily responsible for the binding of ornithine to CPS, while E783 and E892, located within the carbamate domain of the large subunit, were necessary for the transmission of the allosteric signals to the active site. In the K loop for the binding of the monovalent cation potassium, only E761 was crucial for the exhibition of the allosteric effects of ornithine, UMP, and IMP. The mutations H781K and S792K altered significantly the allosteric properties of ornithine, UMP, and IMP, possibly by modifying the conformation of the K-loop structure. Overall, these mutations affected the allosteric properties of ornithine and IMP more than those of UMP. The mutants S792K and D1041A altered the allosteric regulation by ornithine and IMP in a similar way, suggesting common features in the activation mechanism exhibited by these two effectors. © 2002 Elsevier Science (USA) Key Words: Carbamoyl Phosphate synthetase; allosteric control; ornithine. In the companion paper (26), several key amino acids were identified within the allosteric binding site for IMP/UMP from Carbamoyl Phosphate synthetase (CPS) 2 that are responsible for the discrimination and recognition between the nucleotide monoPhosphate effectors. The residues that interact directly with the Phosphate and ribose moieties of IMP had the greatest impact on the binding of the allosteric nucleotide monoPhosphate effectors and on the alterations to the kinetic constants of CPS induced by the binding of either UMP or IMP. One residue, S1016, that hydrogen bonds to the ribose moiety of IMP influenced the binding of IMP more than the binding of UMP, suggesting a potential role in the discrimination between UMP and IMP. However, the residues surrounding the hypoxanthine ring of IMP in the X-ray crystal structure had only a modest influence on the binding constants for the monoPhosphate nucleotides and the allosteric effects exhibited by either UMP or IMP. The largely hydrophobic residues in the IMP binding site, shown to interact with the base of the nucleotide monoPhosphate, appear not to be responsible for the ultimate distinction between UMP and IMP. Ornithine, the first substrate in the biosynthetic pathway for arginine, is an allosteric activator of CPS from Escherichia coli (1-4). Competitive binding experiments have demonstrated that ornithine does not associate physically with the same site as either UMP or IMP (5-7). Ornithine activates CPS by increasing the affinity of the enzyme for the substrate MgATP and this activation phenomenon dominates the inhibitory effects induced by the binding of UMP (6). The specific binding site for the positive allosteric effector ornithine was first identified in the X-ray crystal structure of CPS The concept of a conduit for the propagation of conformational effects from an allosteric ligand binding site to an active site has been formulated for aspartate transCarbamoylase (10 -12) and has been the subject of structure-based computational analyses and molecular 1 To whom correspondence and reprint requests should be ad
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Carbamate Transport in Carbamoyl Phosphate Synthetase: A Theoretical and Experimental Investigation
Journal of the American Chemical Society, 2010Co-Authors: Liliya Lund, Yubo Fan, Qiang Shao, Yi Qin Gao, Frank M. RaushelAbstract: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 ...
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A Combined Theoretical and Experimental Study of the Ammonia Tunnel in Carbamoyl Phosphate Synthetase
Journal of the American Chemical Society, 2009Co-Authors: Yubo Fan, Liliya Lund, Qiang Shao, Yi Qin Gao, Frank M. RaushelAbstract:The transfer of ammonia in Carbamoyl Phosphate synthetase (CPS) was investigated by molecular dynamics simulations and experimental characterization of mutations within the ammonia tunnel. In CPS, ammonia is derived from the hydrolysis of glutamine and this intermediate must travel ∼45 A from the site of formation in the small subunit to the site of utilization in the large subunit. In this investigation, the migration of ammonia was analyzed from the exit of the small subunit through the large subunit where it ultimately reacts with the carboxy Phosphate intermediate. Potential of mean force calculations along the transfer pathway for ammonia indicate a relatively low free-energy barrier for the translocation of ammonia. The highest barrier of 7.2 kcal/mol is found at a narrow turning gate surrounded by the side chains of Cys-232, Ala-251, and Ala-314 in the large subunit. The environment of the ammonia tunnel from the exit of the small subunit to the turning gate in the tunnel is filled with clusters of...
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perforation of the tunnel wall in Carbamoyl Phosphate synthetase derails the passage of ammonia between sequential active sites
Biochemistry, 2004Co-Authors: Jungwook Kim, Frank M. RaushelAbstract:Carbamoyl Phosphate synthetase (CPS) from Escherichia coli consists of a small subunit (∼42 kDa) and a large subunit (∼118 kDa) and catalyzes the biosynthesis of Carbamoyl Phosphate from MgATP, bicarbonate, and glutamine. The enzyme is able to utilize external ammonia as an alternative nitrogen source when glutamine is absent. CPS contains an internal molecular tunnel, which has been proposed to facilitate the translocation of reaction intermediates from one active site to another. Ammonia, the product from the hydrolysis of glutamine in the small subunit, is apparently transported to the next active site in the large subunit of CPS over a distance of about 45 A. The ammonia tunnel that connects these two active sites provides a direct path for the guided diffusion of ammonia and protection from protonation. Molecular damage to the ammonia tunnel was conducted in an attempt to induce leakage of ammonia directly to the protein exterior by the creation of a perforation in the tunnel wall. A hole in the tunn...
David R. Evans - One of the best experts on this subject based on the ideXlab platform.
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aquifex aeolicus aspartate transCarbamoylase an enzyme specialized for the efficient utilization of unstable Carbamoyl Phosphate at elevated temperature
Journal of Biological Chemistry, 2003Co-Authors: Cristina Purcarea, Hedeel I. Guy, Anupama Ahuja, Ladislau C Kovari, David R. EvansAbstract:Aquifex aeolicus, an organism that flourishes at 95 °C, is one of the most thermophilic eubacteria thus far described. The A. aeolicus pyrB gene encoding aspartate transCarbamoylase (ATCase) was cloned, overexpressed in Escherichia coli, and purified by affinity chromatography to a homogeneous form that could be crystallized. Chemical cross-linking and size exclusion chromatography showed that the protein was a homotrimer of 34-kDa catalytic chains. The activity of A. aeolicus ATCase increased dramatically with increasing temperature due to an increase in kcat with little change in the Km for the substrates, Carbamoyl Phosphate and aspartate. The Km for both substrates was 30-40-fold lower than the corresponding values for the homologous E. coli ATCase catalytic subunit. Although rapidly degraded at high temperature, the Carbamoyl Phosphate generated in situ by A. aeolicus Carbamoyl Phosphate synthetase (CPSase) was channeled to ATCase. The transient time for Carbamoyl aspartate formation was 26 s, compared with the much longer transient times observed when A. aeolicus CPSase was coupled to E. coli ATCase. Several other approaches provided strong evidence for channeling and transient complex formation between A. aeolicus ATCase and CPSase. The high affinity for substrates combined with channeling ensures the efficient transfer of Carbamoyl Phosphate from the active site of CPSase to that of ATCase, thus preserving it from degradation and preventing the formation of toxic cyanate.
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a novel Carbamoyl Phosphate synthetase from aquifex aeolicus
Journal of Biological Chemistry, 2001Co-Authors: Anupama Ahuja, Cristina Purcarea, Hedeel I. Guy, David R. EvansAbstract: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.
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Cloning, expression, and structure analysis of carbamate kinase-like Carbamoyl Phosphate synthetase from Pyrococcus abyssi
Extremophiles, 2001Co-Authors: Cristina Purcarea, Guy Hervé, Raymond Cunin, David R. EvansAbstract:Pyrococcus abyssi, a hyperthermophilic archaeon found in the vicinity of deep-sea hydrothermal vents, grows optimally at temperatures around 100°C. Carbamoyl Phosphate synthetase (CPSase) from this organism was cloned and sequenced. The active 34-kDa recombinant protein was overexpressed in Escherichia coli when the host cells were cotransformed with a plasmid encoding tRNA synthetases for low-frequency Escherichia coli codons. Sequence homology suggests that the tertiary structure of P. abyssi CPSase, resembling its counterpart in Pyrococcus furiosus, is closely related to the catabolic carbamate kinases and is very different from the larger mesophilic CPSases. P. furiosus CPSase and carbamate kinase form Carbamoyl Phosphate by phosphorylating carbamate produced spontaneously in solution from ammonia and bicarbonate. In contrast, P. abyssi CPSase has intrinsic bicarbonate-dependent ATPase activity, suggesting that the enzyme can catalyze the phosphorylation of the isosteric substrates carbamate and bicarbonate.
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Regulation of Carbamoyl Phosphate synthetase by MAP kinase.
Nature, 2000Co-Authors: Lee M. Graves, Hedeel I. Guy, Min Huang, Piotr Kozlowski, Matthew Collins, Eduardo R. Lazarowski, R. Marshall Pope, Erik N. Dahlstrand, H. Shelton Earp, David R. EvansAbstract:The de novo synthesis of pyrimidine nucleotides is required for mammalian cells to proliferate. The rate-limiting step in this pathway is catalysed by Carbamoyl Phosphate synthetase (CPS II), part of the multifunctional enzyme CAD1,2. Here we describe the regulation of CAD by the mitogen-activated protein (MAP) kinase cascade. When phosphorylated by MAP kinase in vitro or activated by epidermal growth factor in vivo , CAD lost its feedback inhibition (which is dependent on uridine triPhosphate) and became more sensitive to activation (which depends upon phosphoribosyl pyroPhosphate). Both these allosteric regulatory changes favour biosynthesis of pyrimidines for growth2. They were accompanied by increased epidermal growth factor-dependent phosphorylation of CAD in vivo and were prevented by inhibition of MAP kinase. Mutation of a consensus MAP kinase phosphorylation site abolished the changes in CAD allosteric regulation that were stimulated by growth factors. Finally, consistent with an effect of MAP kinase signalling on CPS II activity, epidermal growth factor increased cellular uridine triPhosphate and this increase was reversed by inhibition of MAP kinase. Hence these studies may indicate a direct link between activation of the MAP kinase cascade and de novo biosynthesis of pyrimidine nucleotides.
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Functional linkage between the glutaminase and synthetase domains of Carbamoyl-Phosphate synthetase. Role of serine 44 in Carbamoyl-Phosphate synthetase-aspartate Carbamoyltransferase-dihydroorotase (cad).
The Journal of biological chemistry, 1999Co-Authors: Anura Hewagama, Hedeel I. Guy, John F. Vickrey, David R. EvansAbstract:Mammalian Carbamoyl-Phosphate synthetase is part of Carbamoyl-Phosphate synthetase-aspartate Carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl Phosphate synthesis requires the concerted action of the glutaminase (GLN) and Carbamoyl-Phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO3−, the other substrates needed for Carbamoyl Phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser44 in the GLN attenuation domain with alanine increases thekcat/Km for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser44 GLN domain and the Escherichia coli Carbamoyl-Phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO3− did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and Carbamoyl Phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser44 → Ala mutant than that needed for Carbamoyl Phosphate synthesis. Ser44, the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and Carbamoyl Phosphate synthesis.
Hazel M. Holden - One of the best experts on this subject based on the ideXlab platform.
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Biological Crystallography The structure of Carbamoyl Phosphate synthetase determined to 2.1 A Ê resolution
2020Co-Authors: James B. Thoden, Frank M. Raushel, Matthew M. Benning, Ivan Rayment, Hazel M. HoldenAbstract:Carbamoyl Phosphate synthetase catalyzes the formation of Carbamoyl Phosphate from one molecule of bicarbonate, two molecules of Mg 2+ 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 ,-heterodimer consisting of a small subunit that hydrolyzes glutamine and a large subunit that catalyzes the two required phosphorylation events. Here the threedimensional structure of Carbamoyl Phosphate synthetase from E. coli re®ned to 2.1 A Ê 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 a-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 Mn 2+ ADP. In the carboxyPhosphate synthetic component, the two observed Mn 2+ 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 Mn 2+ 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
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Long-range allosteric transitions in Carbamoyl Phosphate synthetase.
Protein science : a publication of the Protein Society, 2004Co-Authors: James B. Thoden, Xinyi Huang, Jungwook Kim, Frank M. Raushel, Hazel M. HoldenAbstract: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.
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Carbamoyl Phosphate synthetase creation of an escape route for ammonia
Journal of Biological Chemistry, 2002Co-Authors: James B. Thoden, Xinyi Huang, Frank M. Raushel, Hazel M. HoldenAbstract: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 approximately 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.
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inactivation of the amidotransferase activity of Carbamoyl Phosphate synthetase by the antibiotic acivicin
Journal of Biological Chemistry, 2002Co-Authors: Bryant W. Miles, James B. Thoden, Hazel M. Holden, Frank M. RaushelAbstract:Abstract Carbamoyl Phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of Carbamoyl Phosphate from 2 mol of ATP, bicarbonate, and glutamine. CPS was inactivated by the glutamine analog, acivicin. In the presence of ATP and bicarbonate the second-order rate constant for the inactivation of the glutamine-dependent activities was 4.0 × 104 m −1s−1. In the absence of ATP and bicarbonate the second-order rate constant for inactivation of CPS was reduced by a factor of 200. The enzyme was protected against inactivation by the inclusion of glutamine in the reaction mixture. The ammonia-dependent activities were unaffected by the incubation of CPS with acivicin. These results are consistent with the covalent labeling of the glutamine-binding site located within the small amidotransferase subunit. The binding of ATP and bicarbonate to the large subunit of CPS must also induce a conformational change within the amidotransferase domain of the small subunit that enhances the nucleophilic character of the thiol group required for glutamine hydrolysis. The acivicin-inhibited enzyme was crystallized, and the three-dimensional structure was determined by x-ray diffraction techniques. The thiol group of Cys-269 was covalently attached to the dihydroisoxazole ring of acivicin with the displacement of a chloride ion.
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Carbamoyl Phosphate synthetase an amazing biochemical odyssey from substrate to product
Cellular and Molecular Life Sciences, 1999Co-Authors: Hazel M. Holden, James B. Thoden, Frank M. RaushelAbstract:Carbamoyl Phosphate synthetase (CPS) catalyzes one of the most remarkable reactions ever described in biological chemistry, in which Carbamoyl Phosphate is produced from one molecule of bicarbonate, two molecules of Mg2+ATP, and one molecule of either glutamine or ammonia. The Carbamoyl Phosphate so produced is utilized in the synthesis of arginine and pyrimidine nucleotides. It is also employed in the urea cycle in most terrestrial vertebrates. Due to its large size, its important metabolic role, and the fact that it is highly regulated, CPS has been the focus of intensive investigation for nearly 40 years. Numerous enzymological, biochemical, and biophysical studies by a variety of investigators have led to a quite detailed understanding of CPS. Perhaps one of the most significant advances on this topic within the last 2 years has been the successful X-ray crystallographic analysis of CPS from Escherichia coli. Quite unexpectedly, this structural investigation revealed that the three active sites on the protein are widely separated from one another. Furthermore, these active sites are connected by a molecular tunnel with a total length of approximately 100 A, suggesting that CPS utilizes this channel to facilitate the translocation of reaction intermediates from one site to another. In this review, we highlight the recent biochemical and X-ray crystallographic results that have led to a more complete understanding of this finely tuned instrument of catalysis.
Vicente Rubio - One of the best experts on this subject based on the ideXlab platform.
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structure of human Carbamoyl Phosphate synthetase deciphering the on off switch of human ureagenesis
Scientific Reports, 2015Co-Authors: Vicente Rubio, Javier Cervera, Carmen Diezfernandez, Ignacio Fita, Sergio De Cima, Luis Mariano Polo, Ana MartinezAbstract:Human Carbamoyl Phosphate synthetase (CPS1), a 1500-residue multidomain enzyme, catalyzes the first step of ammonia detoxification to urea requiring N-acetyl-L-glutamate (NAG) as essential activator to prevent ammonia/amino acids depletion. Here we present the crystal structures of CPS1 in the absence and in the presence of NAG, clarifying the on/off-switching of the urea cycle by NAG. By binding at the C-terminal domain of CPS1, NAG triggers long-range conformational changes affecting the two distant phosphorylation domains. These changes, concerted with the binding of nucleotides, result in a dramatic remodeling that stabilizes the catalytically competent conformation and the building of the ~35 A-long tunnel that allows migration of the carbamate intermediate from its site of formation to the second phosphorylation site, where Carbamoyl Phosphate is produced. These structures allow rationalizing the effects of mutations found in patients with CPS1 deficiency (presenting hyperammonemia, mental retardation and even death), as exemplified here for some mutations.
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molecular characterization of Carbamoyl Phosphate synthetase cps1 deficiency using human recombinant cps1 as a key tool
Human Mutation, 2013Co-Authors: Carmen Diezfernandez, Marshall L. Summar, Ana Isabel Martínez, Satu Pekkala, Belén Barcelona, Ana María Guadalajara, Javier Cervera, Isabel Perezarellano, Vicente RubioAbstract:The urea cycle disease Carbamoyl-Phosphate synthetase deficiency (CPS1D) has been associated with many mutations in the CPS1 gene [Haberle et al., 2011. Hum Mutat 32:579-589]. The disease-causing potential of most of these mutations is unclear. To test the mutations effects, we have developed a system for recombinant expression, mutagenesis, and purification of human Carbamoyl-Phosphate synthetase 1 (CPS1), a very large, complex, and fastidious enzyme. The kinetic and molecular properties of recombinant CPS1 are essentially the same as for natural human CPS1. Glycerol partially replaces the essential activator N-acetyl-l-glutamate (NAG), opening possibilities for treating CPS1D due to NAG site defects. The value of our expression system for elucidating the effects of mutations is demonstrated with eight clinical CPS1 mutations. Five of these mutations decreased enzyme stability, two mutations drastically hampered catalysis, and one vastly impaired NAG activation. In contrast, the polymorphisms p.Thr344Ala and p.Gly1376Ser had no detectable effects. Site-limited proteolysis proved the correctness of the working model for the human CPS1 domain architecture generally used for rationalizing the mutations effects. NAG and its analogue and orphan drug N-Carbamoyl-l-glutamate, protected human CPS1 against proteolytic and thermal inactivation in the presence of MgATP, raising hopes of treating CPS1D by chemical chaperoning with N-Carbamoyl-l-glutamate.
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carbamate kinase new structural machinery for making Carbamoyl Phosphate the common precursor of pyrimidines and arginine
Protein Science, 2008Co-Authors: Alberto Marina, Belén Barcelona, Matxalen Uriarte, Ignacio Fita, Pedro M Alzari, Jeronimo Bravo, Vicente RubioAbstract:The enzymes Carbamoyl Phosphate synthetase (CPS) and carbamate kinase (CK) make Carbamoyl Phosphate in the same way: by ATP-phosphorylation of carbamate. The carbamate used by CK is made chemically, whereas CPS itself synthesizes its own carbamate in a process involving the phosphorylation of bicarbonate. Bicarbonate and carbamate are analogs and the phosphorylations are carried out by homologous 40 kDa regions of the 120 kDa CPS polypeptide. CK can also phosphorylate bicarbonate and is a homodimer of a 33 kDa subunit that was believed to resemble the 40 kDa regions of CPS. Such belief is disproven now by the CK structure reported here. The structure does not conform to the biotin carboxylase fold found in the 40 kDa regions of CPS, and presents a new type of fold possibly shared by homologous acylPhosphate-making enzymes. A molecular 16-stranded open β-sheet surrounded by α-helices is the hallmark of the CK dimer. Each subunit also contains two smaller sheets and a large crevice found at the location expected for the active center. Intersubunit interactions are very large and involve a central hydrophobic patch and more hydrophilic peripheral contacts. The crevice holds a sulfate that may occupy the site of an ATP Phosphate, and is lined by conserved residues. Site-directed mutations tested at two of these residues inactivate the enzyme. These findings support active site location in the crevice. The orientation of the crevices in the dimer precludes their physical cooperation in the catalytic process. Such cooperation is not needed in the CK reaction but is a requirement of the mechanism of CPSs.
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understanding Carbamoyl Phosphate synthetase deficiency impact of clinical mutations on enzyme functionality
Journal of Molecular Biology, 2005Co-Authors: Igor Yefimenko, Vicente Rubio, Vicente Fresquet, Clara Marcomarin, Javier CerveraAbstract:Carbamoyl Phosphate synthetase I (CPSI) deficiency, a recessively inherited error of the urea cycle, causes life-threatening hyperammonaemia. CPSI is a multidomain 1500-residue liver mitochondrial matrix protein that is allosterically activated by N-acetyl- l -glutamate, and which synthesises Carbamoyl Phosphate (CP) in three steps: bicarbonate phosphorylation by ATP, carbamate synthesis from carboxyPhosphate and ammonia, and carbamate phosphorylation by ATP. Several missense mutations of CPSI have been reported in patients with CPSI deficiency, but the actual pathogenic potential and effects on the enzyme of these mutations remain non-characterised. Since the structure of Escherichia coli CPS is known and systems for its overexpression and purification are available, we have constructed and purified eight site-directed mutants of E. coli CPS affecting the enzyme large subunit (A126M, R169H, Q262P, N301K, P360L, V640R, R675L, S789P) that are homologous to corresponding missense mutations found in patients with CPSI deficiency, studying their stability and their ability to catalyse the CPS reaction as well as the partial reactions that reflect the different reactional steps, and analysing the substrate kinetics for the overall and partial reactions. The results show that all the mutations significantly decrease CP synthesis without completely inactivating the enzyme (as reflected in the catalysis of at least one partial reaction), that one of these mutations (Q262P) causes marked enzyme instability, and validate the use of E. coli CPS as a pathogenicity testing model for CPSI deficiency. The causality of the reported clinical mutations is supported and the derangements caused by the mutations are identified, revealing the specific roles of the residues that are mutated. In particular, the findings highlight the importance for carbamate phosphorylation and for allosteric activation of a loop that coordinates K+, stress the key role of intersubunit interactions for CPS stability, and suggest that lid opening at both phosphorylation sites is concerted.
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21 Carbamoyl Phosphate synthesis carbamate kinase from pyrococcus furiosus
Methods in Enzymology, 2001Co-Authors: Matxalen Uriarte, Vicente Rubio, Virginie Durbecq, Christianne Legrain, Alberto Marina, Santiago Ramonmaiques, Nicolas GlansdorffAbstract:Publisher Summary Carbamoyl Phosphate (CP) is a precursor of both arginine and the pyrimidines in their de novo biosynthetic pathways. This molecule is highly thermolabile and its thermal decomposition at neutral pH yields cyanate, a nondiscriminate Carbamoylating agent. The metabolism of Carbamoyl Phosphate in hyperthermophilic organisms is therefore of interest. Both carbamate kinase (CK) and Carbamoyl-Phosphate synthase (CPS)can synthesize CP from mixtures of ATP, bicarbonate, and ammonia. The reaction catalyzed by CK is reversible, although the equilibrium favors ATP synthesis. The carbamate kinase described in this article is remarkable by a number of intrinsic properties that are in keeping with the anabolic role it appears to play in vivo in a hyperthermophilic organism: a relatively high affinity for carbamate, a high efficiency in the synthesis of CP as compared to the enteroccocal homolog, and a high thermostability. Moreover, it appears to be engaged in CP channeling with ornithine and aspartate Carbamoyltransferases. The CP-synthesizing activity reported in P. abyssi is probably also because of a true carbamate kinase adapted to anabolism. However, channeling of CP between classical CPS and Carbamoyltransferases specific for ornithine and aspartate has been reported in the extreme thermophilic bacterium Thermus ZO5.
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Cloning, expression, and structure analysis of carbamate kinase-like Carbamoyl Phosphate synthetase from Pyrococcus abyssi
Extremophiles, 2001Co-Authors: Cristina Purcarea, Guy Hervé, Raymond Cunin, David R. EvansAbstract:Pyrococcus abyssi, a hyperthermophilic archaeon found in the vicinity of deep-sea hydrothermal vents, grows optimally at temperatures around 100°C. Carbamoyl Phosphate synthetase (CPSase) from this organism was cloned and sequenced. The active 34-kDa recombinant protein was overexpressed in Escherichia coli when the host cells were cotransformed with a plasmid encoding tRNA synthetases for low-frequency Escherichia coli codons. Sequence homology suggests that the tertiary structure of P. abyssi CPSase, resembling its counterpart in Pyrococcus furiosus, is closely related to the catabolic carbamate kinases and is very different from the larger mesophilic CPSases. P. furiosus CPSase and carbamate kinase form Carbamoyl Phosphate by phosphorylating carbamate produced spontaneously in solution from ammonia and bicarbonate. In contrast, P. abyssi CPSase has intrinsic bicarbonate-dependent ATPase activity, suggesting that the enzyme can catalyze the phosphorylation of the isosteric substrates carbamate and bicarbonate.
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half of saccharomyces cerevisiae Carbamoyl Phosphate synthetase produces and channels Carbamoyl Phosphate to the fused aspartate transCarbamoylase domain
Journal of Biological Chemistry, 1999Co-Authors: Valerie Serre, David R. Evans, Hedeel I. Guy, Bernadette Penverne, Michele Lux, Andrea Rotgeri, Guy HervéAbstract:Abstract The first two steps of the de novopyrimidine biosynthetic pathway in Saccharomyces cerevisiaeare catalyzed by a 240-kDa bifunctional protein encoded by theura2 locus. Although the constituent enzymes, Carbamoyl Phosphate synthetase (CPSase) and aspartate transCarbamoylase (ATCase) function independently, there are interdomain interactions uniquely associated with the multifunctional protein. Both CPSase and ATCase are feedback inhibited by UTP. Moreover, the intermediate Carbamoyl Phosphate is channeled from the CPSase domain where it is synthesized to the ATCase domain where it is used in the synthesis of Carbamoyl aspartate. To better understand these processes, a recombinant plasmid was constructed that encoded a protein lacking the amidotransferase domain and the amino half of the CPSase domain, a 100-kDa chain segment. The truncated complex consisted of the carboxyl half of the CPSase domain fused to the ATCase domain via the pDHO domain, an inactive dihydroorotase homologue that bridges the two functional domains in the native molecule. Not only was the “half CPSase” catalytically active, but it was regulated by UTP to the same extent as the parent molecule. In contrast, the ATCase domain was no longer sensitive to the nucleotide, suggesting that the two catalytic activities are controlled by distinct mechanisms. Most remarkably, isotope dilution and transient time measurements showed that the truncated complex channels Carbamoyl Phosphate. The overall CPSase-ATCase reaction is much less sensitive than the parent molecule to the ATCase bisubstrate analogue,N-phosphonacetyl-l-aspartate (PALA), providing evidence that the endogenously produced Carbamoyl Phosphate is sequestered and channeled to the ATCase active site.
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Channeling of Carbamoyl Phosphate to the Pyrimidine and Arginine Biosynthetic Pathways in the Deep Sea Hyperthermophilic Archaeon Pyrococcus abyssi
Journal of Biological Chemistry, 1999Co-Authors: Cristina Purcarea, David R. Evans, Guy HervéAbstract:The kinetics of the coupled reactions between Carbamoyl-Phosphate synthetase (CPSase) and both aspartate transCarbamoylase (ATCase) and ornithine transCarbamoylase (OTCase) from the deep sea hyperthermophilic archaeon Pyrococcus abyssi demonstrate the existence of Carbamoyl Phosphate channeling in both the pyrimidine and arginine biosynthetic pathways. Isotopic dilution experiments and coupled reaction kinetics analyzed within the context of the formalism proposed by Ovadi et al. (Ovadi, J., Tompa, P., Vertessy, B., Orosz, F., Keleti, T., and Welch, G. R. (1989) Biochem. J. 257, 187-190) are consistent with a partial channeling of the intermediate at 37 degrees C, but channeling efficiency increases dramatically at elevated temperatures. There is no preferential partitioning of Carbamoyl Phosphate between the arginine and pyrimidine biosynthetic pathways. Gel filtration chromatography at high and low temperature and in the presence and absence of substrates did not reveal stable complexes between P. abyssi CPSase and either ATCase or OTCase. Thus, channeling must occur during the dynamic association of coupled enzymes pairs. The interaction of CPSase-ATCase was further demonstrated by the unexpectedly weak inhibition of the coupled reaction by the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA). The anomalous effect of PALA suggests that, in the coupled reaction, the effective concentration of Carbamoyl Phosphate in the vicinity of the ATCase active site is 96-fold higher than the concentration in the bulk phase. Channeling probably plays an essential role in protecting this very unstable intermediate of metabolic pathways performing at extreme temperatures.
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pressure induced dissociation of Carbamoyl Phosphate synthetase domains the catalytically active form is dimeric
Journal of Biological Chemistry, 1998Co-Authors: Hedeel I. Guy, Guy Hervé, Bernard Schmidt, David R. EvansAbstract:Carbamoyl-Phosphate synthetase consists of an amidotransferase domain or subunit (GLN) that hydrolyzes glutamine and transfers the ammonia to the synthetase component (CPS) where the biosynthetic reaction occurs. The CPS domain is composed of two homologous subdomains, CPS.A and CPS.B, that catalyze different ATP-dependent reactions involved in Carbamoyl Phosphate synthesis. When the individual CPS.A and CPS.B subdomains were individually cloned and expressed in Escherichia coli (Guy, H. I., and Evans, D. R. (1996) J. Biol. Chem. 271, 13762-13769), they were found to be functionally equivalent and could each independently catalyze Carbamoyl Phosphate synthesis. The proposal was advanced that, although the monomers could catalyze the individual partial reactions, overall synthesis of Carbamoyl Phosphate required a homodimer of CPS.A or CPS.B. To test this hypothesis, the GLN-CPS.B dimer was reversibly dissociated at 1500 bar in a high pressure cell. Dissociation was accompanied by a loss of both glutamine- and ammonia-dependent CPSase activity. Activity was recovered once the protein was returned to atmospheric pressure. If the sample was cross-linked before exposure to high pressure, there was no dissociation and no loss of biosynthetic activity. In contrast, the bicarbonate-dependent ATPase and the Carbamoyl Phosphate-dependent ATP synthetase activities were largely unaffected by pressure-induced dissociation. These experiments confirmed the hypothesis that the synthesis of Carbamoyl Phosphate requires the concerted action of the two active sites within the homodimer.
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purification and characterization of Carbamoyl Phosphate synthetase from the deep sea hyperthermophilic archaebacterium pyrococcus abyssi
FEBS Journal, 1996Co-Authors: Cristina Purcarea, Daniel Prieur, Valérie Simon, Guy HervéAbstract: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.