The Experts below are selected from a list of 225 Experts worldwide ranked by ideXlab platform
Enoch P Baldwin - One of the best experts on this subject based on the ideXlab platform.
-
crystal structure of escherichia coli cytidine triphosphate synthetase a nucleotide regulated Glutamine Amidotransferase atp dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets
Biochemistry, 2004Co-Authors: James A Endrizzi, Paul M. Anderson, Enoch P BaldwinAbstract:Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and Glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 Glutamine Amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer−tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the Glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the Glutamine hydrolysis active site, providing...
-
crystal structure of escherichia coli cytidine triphosphate synthetase a nucleotide regulated Glutamine Amidotransferase atp dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets
Biochemistry, 2004Co-Authors: James A Endrizzi, Paul M. Anderson, Hanseong Kim, Enoch P BaldwinAbstract:Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and Glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 Glutamine Amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the Glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the Glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics.
James A Endrizzi - One of the best experts on this subject based on the ideXlab platform.
-
crystal structure of escherichia coli cytidine triphosphate synthetase a nucleotide regulated Glutamine Amidotransferase atp dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets
Biochemistry, 2004Co-Authors: James A Endrizzi, Paul M. Anderson, Enoch P BaldwinAbstract:Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and Glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 Glutamine Amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer−tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the Glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the Glutamine hydrolysis active site, providing...
-
crystal structure of escherichia coli cytidine triphosphate synthetase a nucleotide regulated Glutamine Amidotransferase atp dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets
Biochemistry, 2004Co-Authors: James A Endrizzi, Paul M. Anderson, Hanseong Kim, Enoch P BaldwinAbstract:Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and Glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 Glutamine Amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the Glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the Glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics.
Theresa Bridget Fitzpatrick - One of the best experts on this subject based on the ideXlab platform.
-
intersubunit cross talk in pyridoxal 5 phosphate synthase coordinated by the c terminus of the synthase subunit
Journal of Biological Chemistry, 2009Co-Authors: Thomas Raschle, Nikolaus Amrhein, Davide Speziga, Wolfgang Kress, Cyril Moccand, Peter Gehrig, Eilika Weberban, Theresa Bridget FitzpatrickAbstract:Vitamin B(6) is essential in all organisms, due to its requirement as a cofactor in the form of pyridoxal 5'-phosphate (PLP) for key metabolic enzymes. It can be synthesized de novo by either of two pathways known as deoxyxylulose 5-phosphate (DXP)-dependent and DXP-independent. The DXP-independent pathway is the predominant pathway and is found in most microorganisms and plants. A Glutamine Amidotransferase consisting of the synthase Pdx1 and its glutaminase partner, Pdx2, form a complex that directly synthesizes PLP from ribose 5-phosphate, glyceraldehyde 3-phosphate, and Glutamine. The protein complex displays an ornate architecture consisting of 24 subunits, two hexameric rings of 12 Pdx1 subunits to which 12 Pdx2 subunits attach, with the glutaminase and synthase active sites remote from each other. The multiple catalytic ability of Pdx1, the remote glutaminase and synthase active sites, and the elaborate structure suggest regulation of activity on several levels. A missing piece in deciphering this intricate puzzle has been information on the Pdx1 C-terminal region that has thus far eluded structural characterization. Here we use fluorescence spectrophotometry and protein chemistry to demonstrate that the Pdx1 C terminus is indispensable for PLP synthase activity and mediates intersubunit cross-talk within the enzyme complex. We provide evidence that the C terminus can act as a flexible lid, bridging as well as shielding the active site of an adjacent protomer in Pdx1. We show that ribose 5-phosphate binding triggers strong cooperativity in Pdx1, and the affinity for this substrate is substantially enhanced upon interaction with the Michaelis complex of Pdx2 and Glutamine.
-
structure of a bacterial pyridoxal 5 phosphate synthase complex
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Marco Strohmeier, Karsten Rippe, Irmgard Sinning, Theresa Bridget Fitzpatrick, Thomas Raschle, Jacek Mazurkiewicz, Ivo TewsAbstract:Vitamin B6 is an essential metabolic cofactor that has more functions in humans than any other single nutrient. Its de novo biosynthesis occurs through two mutually exclusive pathways that are absent in animals. The predominant pathway found in most prokaryotes, fungi, and plants has only recently been discovered. It is distinguished by a Glutamine Amidotransferase, which is remarkable in that it alone can synthesize the cofactor form, pyridoxal 5'-phosphate (PLP), directly from a triose and a pentose saccharide and Glutamine. Here we report the 3D structure of the PLP synthase complex with substrate Glutamine bound as well as those of the individual synthase and glutaminase subunits Pdx1 and Pdx2, respectively. The complex is made up of 24 protein units assembled like a cogwheel, a dodecameric Pdx1 to which 12 Pdx2 subunits attach. In contrast to the architecture of previously determined Glutamine Amidotransferases, macromolecular assembly is directed by an N-terminal alpha-helix on the synthase. Interaction with the synthase subunit leads to glutaminase activation, resulting in formation of an oxyanion hole, a prerequisite for catalysis. Mutagenesis permitted identification of the remote glutaminase and synthase catalytic centers and led us to propose a mechanism whereby ammonia shuttles between these active sites through a methionine-rich hydrophobic tunnel.
-
vitamin b6 biosynthesis by the malaria parasite plasmodium falciparum biochemical and structural insights
Journal of Biological Chemistry, 2006Co-Authors: Martin Gengenbacher, Irmgard Sinning, Ivo Tews, Theresa Bridget Fitzpatrick, Thomas Raschle, Karlheinz Flicker, Sylke Muller, Peter Macheroux, Barbara KappesAbstract:Vitamin B6 is one of nature's most versatile cofactors. Most organisms synthesize vitamin B6 via a recently discovered pathway employing the proteins Pdx1 and Pdx2. Here we present an in-depth characterization of the respective orthologs from the malaria parasite, Plasmodium falciparum. Expression profiling of Pdx1 and -2 shows that blood-stage parasites indeed possess a functional vitamin B6 de novo biosynthesis. Recombinant Pdx1 and Pdx2 form a complex that functions as a Glutamine Amidotransferase with Pdx2 as the glutaminase and Pdx1 as pyridoxal-5 '-phosphate synthase domain. Complex formation is required for catalytic activity of either domain. Pdx1 forms a chimeric bi-enzyme with the bacterial YaaE, a Pdx2 ortholog, both in vivo and in vitro, although this chimera does not attain full catalytic activity, emphasizing that species-specific structural features govern the interaction between the protein partners of the PLP synthase complexes in different organisms. To gain insight into the activation mechanism of the parasite bi-enzyme complex, the three-dimensional structure of Pdx2 was determined at 1.62 A. The obstruction of the oxyanion hole indicates that Pdx2 is in a resting state and that activation occurs upon Pdx1-Pdx2 complex formation.
-
on the two components of pyridoxal 5 phosphate synthase from bacillus subtilis
Journal of Biological Chemistry, 2005Co-Authors: Thomas Raschle, Nikolaus Amrhein, Theresa Bridget FitzpatrickAbstract:Vitamin B6 is an essential nutrient in the human diet. It can act as a co-enzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant. Plants and microorganisms have the ability to make the compound. Yet, studies of vitamin B6 biosynthesis have been mainly restricted to Escherichia coli, where the vitamin is synthesized from 1-deoxy-d -xylulose 5-phosphate and 4-phosphohydroxy-l-threonine. Recently, a novel pathway for its synthesis has been discovered, involving two genes (PDX1 and PDX2) neither of which is homologous to any of those participating in the E. coli pathway. In Bacillus subtilis, YaaD and YaaE represent the PDX1 and PDX2 homolog, respectively. The two proteins form a complex that functions as a Glutamine Amidotransferase, with YaaE as the glutaminase domain and YaaD as the acceptor and pyridoxal 5'-phosphate (PLP) synthesis domain. In this report we corroborate a recent report on the identification of the substrates of YaaD and provide unequivocal proof of the identity of the reaction product. We show that both the glutaminase and synthase reactions are dependent on the respective protein partner. The synthase reaction can also utilize an external ammonium source but, in contrast to other Glutamine Amidotransferases, is dependent on YaaE under certain conditions. Furthermore, we report on the detailed characterization of the inhibition of the glutaminase domain, and thus PLP synthesis, by the Glutamine analog acivicin. Employing pull-out assays and native-PAGE, we provide evidence for the dissociation of the bi-enzyme complex under these conditions. The results are discussed in light of the nature of the interaction of the two components of the enzyme complex.
Janet L Smith - One of the best experts on this subject based on the ideXlab platform.
-
crystal structures capture three states in the catalytic cycle of a pyridoxal phosphate plp synthase
Journal of Biological Chemistry, 2015Co-Authors: Amber Marie Smith, Janet L Smith, William Clay Brown, E HarmsAbstract:Abstract PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5′-phosphate (PLP) from Glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine. The first intermediate forms with ribose 5-phosphate. The glutaminase subunit is a Glutamine Amidotransferase that hydrolyzes Glutamine and channels ammonia to the synthase active site. Ammonia attack on the first covalent intermediate forms the second intermediate. Glyceraldehyde 3-phosphate reacts with the second intermediate to form PLP. To investigate the mechanism of the synthase subunit, crystal structures were obtained for three intermediate states of the Geobacillus stearothermophilus intact PLPS or its synthase subunit. The structures capture the synthase active site at three distinct steps in its complicated catalytic cycle, provide insights into the elusive mechanism, and illustrate the coordinated motions within the synthase subunit that separate the catalytic states. In the intact PLPS with a Michaelis-like intermediate in the glutaminase active site, the first covalent intermediate of the synthase is fully sequestered within the enzyme by the ordering of a generally disordered 20-residue C-terminal tail. Following addition of ammonia, the synthase active site opens and admits the Lys-149 side chain, which participates in formation of the second intermediate and PLP. Roles are identified for conserved Asp-24 in the formation of the first intermediate and for conserved Arg-147 in the conversion of the first to the second intermediate.
-
toward understanding the mechanism of the complex cyclization reaction catalyzed by imidazole glycerolphosphate synthase crystal structures of a ternary complex and the free enzyme
Biochemistry, 2003Co-Authors: Barnali N Chaudhuri, Rebecca S. Myers, Jo V Davisson, Stephanie C Lange, Janet L SmithAbstract:Imidazole glycerol phosphate synthase catalyzes formation of the imidazole ring in histidine biosynthesis. The enzyme is also a Glutamine Amidotransferase, which produces ammonia in a glutaminase active site and channels it through a 30-A internal tunnel to a cyclase active site. Glutaminase activity is impaired in the resting enzyme, and stimulated by substrate binding in the cyclase active site. The signaling mechanism was investigated in the crystal structure of a ternary complex in which the glutaminase active site was inactivated by a Glutamine analogue and the unstable cyclase substrate was cryo-trapped in the active site. The orientation of N1-(5‘-phosphoribulosyl)-formimino-5-aminoimidazole-4-carboxamide ribonucleotide in the cyclase active site implicates one side of the cyclase domain in signaling to the glutaminase domain. This side of the cyclase domain contains the interdomain hinge. Two interdomain hydrogen bonds, which do not exist in more open forms of the enzyme, are proposed as molecular...
-
coupled formation of an Amidotransferase interdomain ammonia channel and a phosphoribosyltransferase active site
Biochemistry, 1997Co-Authors: Joseph M Krahn, Jeong Hyun Kim, Mark R Burns, Ronald J Parry, Howard Zalkin, Janet L SmithAbstract:Activation of gluatmine phosphoribosylpyrophosphate (RPPP) Amidotransferase (GPATase) by binding of a PRPP substrate analog results in the formation of a 20 A channel connecting the active site for Glutamine hydrolysis in one domain with the PRPP site in a second domain. This solvent-inaccessible channel permits transfer of the NH3 intermediate between the two active sites. Tunneling of NH3 may be a common mechanism for Glutamine Amidotransferase-catalyzed nitrogen transfer and for coordination of catalysis at two distinct active sites in complex enzymes. The 2.4 A crystal structure of the active conformer of GPATase also provides the first description of an intact active site for the phosphoribosyltransferase (PRTase) family of nucleotide synthesis and salvage enzymes. Chemical assistance to catalysis is provided primarily by the substrate and secondarily by the enzyme in the proposed structure-based mechanism. Different catalytic and inhibitory modes of divalent cation binding to the PRTase active site are revealed in the active conformer of the enzyme and in a feedback-inhibited GMP complex.
-
preliminary x ray analysis of escherichia coli gmp synthetase determination of anomalous scattering factors for a cysteinyl mercury derivative
Proteins, 1994Co-Authors: John J G Tesmer, Timothy L Stemmler, James E Pennerhahn, Jo V Davisson, Janet L SmithAbstract:We have initiated a project to determine the three-dimensional structure of GMP synthetase (GMPS) from Escherichia coli. GMPS catalyzes the conversion of XMP to GMP in the final step of de novo guanine nucleotide biosynthesis, and is a member of the Glutamine Amidotransferase family: a group of enzymes responsible for the assimilation of nitrogen into compounds such as amino acids, purine and pyrimidine bases, amino sugars, and antibiotics. The E. coli guaA gene encoding GMPS was cloned into a tac expression vector, overexpressed, and its gene product purified. Conditions for the growth of protein crystals were developed using recombinant GMPS in the presence of MgCl2, ATP, and XMP. The crystals are monoclinic, space group P2(1), with cell parameters of a = 156.0 A, b = 102.0 A, c = 78.8 A, beta = 96.7 degrees. Diffraction data to 2.8 A spacings were collected on a Xuong-Hamlin area detector with an overall Rsym of 5.2%. Both the volume of the unit cell and the peaks in the self-rotation function are consistent with one GMPS tetramer of D2 symmetry in the crystallographic asymmetric unit. Previously, GMPS has been observed only as a dimer in solution. GMPS was covalently modified with p-chloromercuribenzylsulfonic acid (PCMBS), and its X-ray fluorescence spectrum was measured through the LIII absorption edge of mercury. Anomalous scattering factors for cysteinyl mercury were derived from this spectrum, and the feasibility of structure determination by multi-wavelength anomalous diffraction was evaluated. The optimal MAD dispersive signal is 4.5% of magnitude of F, and the optimal MAD Bijvoet signal is 7.5% of magnitude of F at a concentration of approximately 1 mercury per 10-kDa protein. The anomalous scattering factors tabulated here should be transferable to cysteinyl mercury in other proteins.
Paul M. Anderson - One of the best experts on this subject based on the ideXlab platform.
-
crystal structure of escherichia coli cytidine triphosphate synthetase a nucleotide regulated Glutamine Amidotransferase atp dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets
Biochemistry, 2004Co-Authors: James A Endrizzi, Paul M. Anderson, Enoch P BaldwinAbstract:Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and Glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 Glutamine Amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer−tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the Glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the Glutamine hydrolysis active site, providing...
-
crystal structure of escherichia coli cytidine triphosphate synthetase a nucleotide regulated Glutamine Amidotransferase atp dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets
Biochemistry, 2004Co-Authors: James A Endrizzi, Paul M. Anderson, Hanseong Kim, Enoch P BaldwinAbstract:Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and Glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 Glutamine Amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the Glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the Glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics.