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Bernd Nidetzky - One of the best experts on this subject based on the ideXlab platform.
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interplay of catalytic subsite residues in the positioning of α d Glucose 1 Phosphate in sucrose phosphorylase
Biochemistry and biophysics reports, 2015Co-Authors: Patricia Wildberger, Lothar Brecker, Gaia A Aish, David L Jakeman, Bernd NidetzkyAbstract:Kinetic and molecular docking studies were performed to characterize the binding of α-d-Glucose 1-Phosphate (αGlc 1-P) at the catalytic subsite of a family GH-13 sucrose phosphorylase (from L. mesenteroides) in wild-type and mutated form. The best-fit binding mode of αGlc 1-P dianion had the Phosphate group placed anti relative to the glucosyl moiety (adopting a relaxed 4C1 chair conformation) and was stabilized mainly by hydrogen bonds from residues of the enzyme׳s catalytic triad (Asp196, Glu237 and Asp295) and from Arg137. Additional feature of the αGlc 1-P docking pose was an intramolecular hydrogen bond (2.7 A) between the glucosyl C2-hydroxyl and the Phosphate oxygen. An inactive phosphonate analog of αGlc 1-P did not show binding to sucrose phosphorylase in different experimental assays (saturation transfer difference NMR, steady-state reversible inhibition), consistent with evidence from molecular docking study that also suggested a completely different and strongly disfavored binding mode of the analog as compared to αGlc 1-P. Molecular docking results also support kinetic data in showing that mutation of Phe52, a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase. However, when combined with a second mutation involving one of the catalytic triad residues, the mutation of Phe52 by Ala caused complete (F52A_D196A; F52A_E237A) or very large (F52A_D295A) disruption of the proposed productive binding mode of αGlc 1-P with consequent effects on the enzyme activity. Effects of positioning of αGlc 1-P for efficient glucosyl transfer from Phosphate to the catalytic nucleophile of the enzyme (Asp196) are suggested. High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.
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phosphoryl transfer from α d Glucose 1 Phosphate catalyzed by escherichia coli sugar Phosphate phosphatases of two protein superfamily types
Applied and Environmental Microbiology, 2015Co-Authors: Patricia Wildberger, Lothar Brecker, Martin Pfeiffer, Gerald N Rechberger, Ruth Birnergruenberger, Bernd NidetzkyAbstract:The Cori ester α-d-Glucose 1-Phosphate (αGlc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes αGlc 1-P for flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-Phosphate phosphatases from Escherichia coli were characterized in this study for utilization of αGlc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic αGlc 1-P phosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (in E. coli Origami B) gave a functional enzyme preparation (kcat for phosphoryl transfer from αGlc 1-P to water, 40 s−1) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys189 and Cys195. Enzymatic phosphoryl transfer from αGlc 1-P to water in H218O solvent proceeded with complete 18O label incorporation into the Phosphate released, consistent with catalytic reaction through O-1–P, but not C-1–O, bond cleavage. Hydrolase activity of both enzymes was not restricted to a glycosidic phosphomonoester substrate, and d-Glucose 6-Phosphate was converted with a kcat similar to that of αGlc 1-P. By examining phosphoryl transfer from αGlc 1-P to an acceptor substrate other than water (d-fructose or d-Glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized αGlc 1-P mainly for phosphoryl transfer to water. By applying d-fructose in 10-fold molar excess over αGlc 1-P (20 mM), enzymatic conversion furnished d-fructose 1-Phosphate as the main product in a 55% overall yield. Agp is a promising biocatalyst for use in transphosphorylation from αGlc 1-P.
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yihx encoded haloacid dehalogenase like phosphatase had4 from escherichia coli is a specific α d Glucose 1 Phosphate hydrolase useful for substrate selective sugar Phosphate transformations
Journal of Molecular Catalysis B-enzymatic, 2014Co-Authors: Martin Pfeiffer, Patricia Wildberger, Bernd NidetzkyAbstract:Phosphomonoester hydrolases (phosphatases; EC 3.1.3.) often exhibit extremely relaxed substrate specificity which limits their application to substrate-selective biotransformations. In search of a phosphatase catalyst specific for hydrolyzing α-d-Glucose 1-Phosphate (αGlc 1-P), we selected haloacid dehalogenase-like phosphatase 4 (HAD4) from Escherichia coli and obtained highly active recombinant enzyme through a fusion protein (Zbasic2_HAD4) that contained Zbasic2, a strongly positively charged three α-helical bundle module, at its N-terminus. Highly pure Zbasic2_HAD4 was prepared directly from E. coli cell extract using capture and polishing combined in a single step of cation exchange chromatography. Kinetic studies showed Zbasic2_HAD4 to exhibit 565-fold preference for hydrolyzing αGlc 1-P (kcat/KM = 1.87 ± 0.03 mM−1 s−1; 37 °C, pH 7.0) as compared to d-Glucose 6-Phosphate (Glc 6-P). Also among other sugar Phosphates, αGlc 1-P was clearly preferred. Using different mixtures of αGlc 1-P and Glc 6-P (e.g. 180 mM each) as the substrate, Zbasic2_HAD4 could be used to selectively convert the αGlc 1-P present, leaving back all of the Glc 6-P for recovery. Zbasic2_HAD4 was immobilized conveniently using direct loading of E. coli cell extract on sulfonic acid group-containing porous carriers, yielding a recyclable heterogeneous biocatalyst that was nearly as effective as the soluble enzyme, probably because protein attachment to the anionic surface occurred in a preferred orientation via the cationic Zbasic2 module. Selective removal of αGlc 1-P from sugar Phosphate preparations could be an interesting application of Zbasic2_HAD4 for which readily available broad-spectrum phosphatases are unsuitable.
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examining the role of Phosphate in glycosyl transfer reactions of cellulomonas uda cellobiose phosphorylase using d glucal as donor substrate
Carbohydrate Research, 2012Co-Authors: Patricia Wildberger, Lothar Brecker, Bernd NidetzkyAbstract:Abstract Cellobiose phosphorylase from Cellulomonas uda ( Cu CPase) is shown to utilize d -glucal as slow alternative donor substrate for stereospecific glycosyl transfer to inorganic Phosphate, giving 2-deoxy-α- d -Glucose 1-Phosphate as the product. When performed in D 2 O, enzymatic phosphorolysis of d -glucal proceeds with incorporation of deuterium in equatorial position at C-2, implying a stereochemical course of reaction where substrate becomes protonated from below its six-membered ring through stereoselective re side attack at C-2. The proposed catalytic mechanism, which is supported by results of docking studies, involves direct protonation of d -glucal by the enzyme-bound Phosphate, which then performs nucleophilic attack on the reactive C-1 of donor substrate. When offered d -Glucose next to d -glucal and Phosphate, Cu CPase produces 2-deoxy-β- d -glucosyl-(1→4)- d -Glucose and 2-deoxy-α- d -Glucose 1-Phosphate in a ratio governed by mass action of the two acceptor substrates present. Enzymatic synthesis of 2-deoxy-β- d -glucosyl-(1→4)- d -Glucose is effectively promoted by catalytic concentrations of Phosphate, suggesting that catalytic reaction proceeds through a quaternary complex of Cu CPase, d -glucal, Phosphate, and d -Glucose. Conversion of d -glucal and Phosphate presents a convenient single-step synthesis of 2-deoxy-α- d -Glucose 1-Phosphate that is difficult to prepare chemically.
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recombinant sucrose phosphorylase from leuconostoc mesenteroides characterization kinetic studies of transglucosylation and application of immobilised enzyme for production of α d Glucose 1 Phosphate
Journal of Biotechnology, 2007Co-Authors: Christiane Goedl, Alexandra Schwarz, Alphonse Minani, Bernd NidetzkyAbstract:Abstract Sucrose phosphorylase catalyzes the reversible conversion of sucrose (α- d -glucopyranosyl-1,2-β- d -fructofuranoside) and Phosphate into d -fructose and α- d -Glucose 1-Phosphate. We report on the molecular cloning and expression of the structural gene encoding sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase) in Escherichia coli DH10B. The recombinant enzyme, containing an 11 amino acid-long N-terminal metal affinity fusion peptide, was overproduced 60-fold in comparison with the natural enzyme. It was purified to apparent homogeneity using copper-loaded Chelating Sepharose and obtained in 20% yield with a specific activity of 190 U mg−1. LmSPase was covalently attached onto Eupergit C with a binding efficiency of 50% and used for the continuous production of α- d -Glucose 1-Phosphate from sucrose and Phosphate (600 mM each) in a packed-bed immobilised enzyme reactor (30 °C, pH 7.0). The reactor was operated at a stable conversion of 91% (550 mM product) and productivity of approximately 11 g l−1 h−1 for up to 600 h. A kinetic study of transglucosylation by soluble LmSPase was performed using α- d -Glucose 1-Phosphate as the donor substrate and various alcohols as acceptors. d - and l -arabitol were found to be good glucosyl acceptors.
James H. Naismith - One of the best experts on this subject based on the ideXlab platform.
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next generation Glucose 1 Phosphate thymidylyltransferase rmla inhibitors an extended sar study to direct future design
Bioorganic & Medicinal Chemistry, 2021Co-Authors: Ganyuan Xiao, Magnus S Alphey, Lisa Pirrie, Fanny Tran, Pierre Milbeo, Yi Zhou, Jasmine Bickel, Oxana Kempf, Karl Kempf, James H. NaismithAbstract:The monosaccharide l-Rhamnose is an important component of bacterial cell walls. The first step in the l-rhamnose biosynthetic pathway is catalysed by Glucose-1-Phosphate thymidylyltransferase (RmlA), which condenses Glucose-1-Phosphate (Glu-1-P) with deoxythymidine triPhosphate (dTTP) to yield dTDP-d-Glucose. In addition to the active site where catalysis of this reaction occurs, RmlA has an allosteric site that is important for its function. Building on previous reports, SAR studies have explored further the allosteric site, leading to the identification of very potent P. aeruginosa RmlA inhibitors. Modification at the C6-NH2 of the inhibitor's pyrimidinedione core structure was tolerated. X-ray crystallographic analysis of the complexes of P. aeruginosa RmlA with the novel analogues revealed that C6-aminoalkyl substituents can be used to position a modifiable amine just outside the allosteric pocket. This opens up the possibility of linking a siderophore to this class of inhibitor with the goal of enhancing bacterial cell wall permeability.
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the structural basis of the catalytic mechanism and regulation of Glucose 1 Phosphate thymidylyltransferase rmla
The EMBO Journal, 2000Co-Authors: Wulf Blankenfeldt, Joseph S. Lam, Miryam Asuncion, James H. NaismithAbstract:The synthesis of deoxy-thymidine di-Phosphate (dTDP)-L-rhamnose, an important component of the cell wall of many microorganisms, is a target for therapeutic intervention. The first enzyme in the dTDP-L-rhamnose biosynthetic pathway is Glucose-1-Phosphate thymidylyltransferase (RmlA). RmlA is inhibited by dTDP-L-rhamnose thereby regulating L-rhamnose production in bacteria. The structure of Pseudomonas aeruginosa RmlA has been solved to 1.66 A resolution. RmlA is a homotetramer, with the monomer consisting of three functional subdomains. The sugar binding and dimerization subdomains are unique to RmlA-like enzymes. The sequence of the core subdomain is found not only in sugar nucleotidyltransferases but also in other nucleotidyltransferases. The structures of five distinct enzyme substrate- product complexes reveal the enzyme mechanism that involves precise positioning of the nucleophile and activation of the electrophile. All the key residues are within the core subdomain, suggesting that the basic mechanism is found in many nucleotidyltransferases. The dTDP-L-rhamnose complex identifies how the protein is controlled by its natural inhibitor. This work provides a platform for the design of novel drugs against pathogenic bacteria.
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The purification, crystallization and preliminary structural characterization of Glucose-1-Phosphate thymidylyltransferase (RmlA), the first enzyme of the dTDP-L-rhamnose synthesis pathway from Pseudomonas aeruginosa.
Acta Crystallographica Section D Biological Crystallography, 2000Co-Authors: Wulf Blankenfeldt, Marie-france Giraud, Gordon A. Leonard, Rahim Rahim, Carole Creuzenet, Joseph S. Lam, James H. NaismithAbstract:Glucose-1-Phosphate thymidylyltransferase (RmlA; E.C. 2.7.7.24) is the first of four enzymes involved in the biosynthesis of dTDP-l-rhamnose, the precursor of l-rhamnose, a key component of the cell wall of many pathogenic bacteria. RmlA catalyses the condensation of thymidine triPhosphate (dTTP) and α-d-Glucose-1-Phosphate (G1P), yielding dTDP-d-Glucose. RmlA from Pseudomonas aeruginosa has been overexpressed and purified. Crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with PEG 6000 and lithium sulfate as precipitant. Several diffraction data sets of single frozen crystals were collected to a resolution of 1.66 A. Crystals belonged to space group P1, with unit-cell parameters a = 71.5, b = 73.1, c = 134.7 A, α = 89.9, β = 80.9, γ = 81.1°. The asymmetric unit contains eight monomers in the form of two RmlA tetramers with a solvent content of 51%. Selenomethionine-labelled protein has been obtained and crystallized.
Hazel M. Holden - One of the best experts on this subject based on the ideXlab platform.
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the structure of Glucose 1 Phosphate thymidylyltransferase from mycobacterium tuberculosis reveals the location of an essential magnesium ion in the rmla type enzymes
Protein Science, 2018Co-Authors: Haley A Brown, Peter A Tipton, James B. Thoden, Hazel M. HoldenAbstract:Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, continues to be a major threat to populations worldwide. Whereas the disease is treatable, the drug regimen is arduous at best with the use of four antimicrobials over a six-month period. There is clearly a pressing need for the development of new therapeutics. One potential target for structure-based drug design is the enzyme RmlA, a Glucose-1-Phosphate thymidylyltransferase. This enzyme catalyzes the first step in the biosynthesis of l-rhamnose, which is a deoxysugar critical for the integrity of the bacterium's cell wall. Here we report the X-ray structures of M. tuberculosis RmlA in complex with either dTTP or dTDP-Glucose to 1.6 A and 1.85 A resolution, respectively. In the RmlA/dTTP complex, two magnesium ions were observed binding to the nucleotide, both ligated in octahedral coordination spheres. In the RmlA/dTDP-Glucose complex, only a single magnesium ion was observed. Importantly, for RmlA-type enzymes with known three-dimensional structures, not one model shows the position of the magnesium ion bound to the nucleotide-linked sugar. As such, this investigation represents the first direct observation of the manner in which a magnesium ion is coordinated to the RmlA product and thus has important ramifications for structure-based drug design. In the past, molecular modeling procedures have been employed to derive a three-dimensional model of the M. tuberculosis RmlA for drug design. The X-ray structures presented herein provide a superior molecular scaffold for such endeavors in the treatment of one of the world's deadliest diseases. This article is protected by copyright. All rights reserved.
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Active site geometry of Glucose-1-Phosphate uridylyltransferase.
Protein science : a publication of the Protein Society, 2007Co-Authors: James B. Thoden, Hazel M. HoldenAbstract:Glucose-1-Phosphate uridylyltransferase, or UGPase, catalyzes the production of UDP-Glucose from Glucose-1-Phosphate and UTP. Because of the biological role of UDP-Glucose in glycogen synthesis and in the formation of glycolipids, glycoproteins, and proteoglycans, the enzyme is widespread in nature. Recently this laboratory reported the three-dimensional structure of UGPase from Escherichia coli. While the initial X-ray analysis revealed the overall fold of the enzyme, details concerning its active site geometry were limited because crystals of the protein complexed with either substrates or products could never be obtained. In an effort to more fully investigate the active site geometry of the enzyme, UGPase from Corynebacterium glutamicum was subsequently cloned and purified. Here we report the X-ray structure of UGPase crystallized in the presence of both magnesium and UDP-Glucose. Residues involved in anchoring the ligand to the active site include the polypeptide chain backbone atoms of Ala 20, Gly 21, Gly 117, Gly 180, and Ala 214, and the side chains of Glu 36, Gln 112, Asp 143, Glu 201, and Lys 202. Two magnesium ions are observed coordinated to the UDP-Glucose. An α- and a β-phosphoryl oxygen, three waters, and the side chain of Asp 142 ligate the first magnesium, whereas the second ion is coordinated by an α-phosphoryl oxygen and five waters. The position of the first magnesium is conserved in both the Glucose-1-Phosphate thymidylyltransferases and the cytidylyltransferases. The structure presented here provides further support for the role of the conserved magnesium ion in the catalytic mechanisms of the sugar-1-Phosphate nucleotidylyltransferases.
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The molecular architecture of Glucose-1-Phosphate uridylyltransferase
Protein science : a publication of the Protein Society, 2007Co-Authors: James B. Thoden, Hazel M. HoldenAbstract:Glucose-1-Phosphate uridylyltransferase, also referred to as UDP-Glucose pyrophosphorylase or UGPase, catalyzes the formation of UDP-Glucose from Glucose-1-Phosphate and UTP. Not surprisingly, given the central role of UDP-Glucose in glycogen synthesis and in the production of glycolipids, glycoproteins, and proteoglycans, the enzyme is ubiquitous in nature. Interestingly, however, the prokaryotic and eukaryotic forms of the enzyme are unrelated in amino acid sequence and structure. Here we describe the cloning and structural analysis to 1.9 Aresolution of the UGPase from Escherichia coli. The protein is a tetramer with 222 point group symmetry. Each subunit of the tetramer is dominated by an eight-stranded mixed b-sheet. There are two additional layers of b-sheet (two and three strands) and 10 a-helices. The overall fold of the molecule is remarkably similar to that observed for Glucose-1- Phosphate thymidylyltransferase in complex with its product, dTDP-Glucose. On the basis of this similarity, a UDP-Glucose moiety has been positioned into the active site of UGPase. This protein/product model predicts that the side chains of Gln 109 and Asp 137, respectively, serve to anchor the uracil ring and the ribose of UDP-Glucose to the protein. The b-phosphoryl group of the product is predicted to lie within hydrogen bonding distance to the e-nitrogen of Lys 202 whereas the carboxylate group of Glu 201 is predicted to bridge the 29 -a nd 39-hydroxyl groups of the glucosyl moiety. Details concerning the overall structure of UGPase and a comparison with Glucose-1-Phosphate thymidylyltransferase are presented.
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kinetic and structural analysis of α d Glucose 1 Phosphate cytidylyltransferase from salmonella typhi
Journal of Biological Chemistry, 2005Co-Authors: Nicole M. Koropatkin, Wallace W Cleland, Hazel M. HoldenAbstract:Tyvelose is a 3,6-dideoxyhexose found in the O-antigen of the surface lipopolysaccharides of some pathogenic bacteria. It is synthesized via a complex biochemical pathway that is initiated by the formation of CDP-D-Glucose. The production of this ligand is catalyzed by the enzyme Glucose-1-Phosphate cytidylyltransferase, which utilizes alpha-D-Glucose 1-Phosphate and MgCTP as substrates. Previous x-ray crystallographic investigations have demonstrated that the Salmonella typhi enzyme complexed with the product CDP-Glucose is a fully integrated hexamer displaying 32 point group symmetry. The binding pocket for CDP-Glucose is shared between two subunits. Here we describe both a detailed kinetic analysis of the cytidylyltransferase and a structural investigation of the enzyme complexed with MgCTP. These data demonstrate that the reaction catalyzed by the cytidylyltransferase proceeds via a sequential rather than a Bi Bi ping-pong mechanism as was previously reported. Additionally, the enzyme utilizes both CTP and UTP equally well as substrates. The structure of the enzyme with bound MgCTP reveals that the binding pocket for the nucleotide is contained within one subunit rather than shared between two. Key side chains involved in nucleotide binding include Thr(14), Arg(15), Lys(25), and Arg(111). In the previous structure of the enzyme complexed with CDP-Glucose, those residues defined by Thr(14) to Ile(21) were disordered. The kinetic and x-ray crystallographic data presented here support a mechanism for this enzyme that is similar to that reported for the Glucose-1-Phosphate thymidylyltransferases.
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Molecular Structure of α-d-Glucose-1-Phosphate Cytidylyltransferase from Salmonella typhi
The Journal of biological chemistry, 2004Co-Authors: Nicole M. Koropatkin, Hazel M. HoldenAbstract:Abstract Dideoxysugars, which display biological activities ranging from mediating cell-cell interactions to serving as components in some antibiotics, are synthesized in various organisms via complex biochemical pathways that begin with the attachment of α-d-Glucose 1-Phosphate to either CTP or dTTP. Here we describe the three-dimensional structure of the α-d-Glucose-1-Phosphate cytidylyltransferase from Salmonella typhi, which catalyzes the first step in the production of CDP-tyvelose. For this investigation, the enzyme was crystallized in the presence of its product, CDP-Glucose. In contrast to previous reports, the enzyme exists as a fully integrated hexamer with 32-point group symmetry. Each subunit displays a “bird-like” appearance with the “body” composed predominantly of a seven-stranded mixed β-sheet and the two “wings” formed by β-hairpin motifs. These two wings mediate subunit-subunit interactions along the 3-fold and 2-fold rotational axes, respectively. The six active sites of the hexamer are situated between the subunits related by the 2-fold rotational axes. CDP-Glucose is anchored to the protein primarily by hydrogen bonds with backbone carbonyl oxygens and peptidic NH groups. The side chains of Arg111 and Asn188 from one subunit and Glu178 and Lys179 from the second subunit are also involved in hydrogen bonding with the ligand. The topology of the main core domain bears striking similarity to that observed for Glucose-1-Phosphate thymidylyltransferase and 4-diphosphocytidyl-2-C-methylerythritol synthetase.
Norio Shiomi - One of the best experts on this subject based on the ideXlab platform.
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structural analysis of six novel oligosaccharides synthesized by glucosyl transfer from β d Glucose 1 Phosphate to raffinose and stachyose using thermoanaerobacter brockii kojibiose phosphorylase
Tetrahedron-asymmetry, 2005Co-Authors: Natsuko Takahashi, Tomoyuki Nishimoto, Hideki Okada, Eri Fukushi, Shuichi Onodera, Jun Kawabata, Norio ShiomiAbstract:Abstract Novel oligosaccharides (one tetra-, two penta-, two hexa- and one hepta-saccharides) were synthesized by glucosyl transfer from β- d -Glucose 1-Phosphate (β- d -G1P) to raffinose or stachyose using Thermoanaerobacter brockii kojibiose phosphorylase. Gas liquid chromatography analysis of methyl derivatives, MALDI-TOF-MS and NMR measurements were used for structural confirmation. The 1 H and 13 C NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, HMBC and CH 2 -selected E-HSQC techniques. These oligosaccharides were identified as 2-α- d -glucopyranosyl-raffinose, 2 G (2-α- d -glucopyranosyl) 2 -raffinose, 2 G (2-α- d -glucopyranosyl) 3 -raffinose, 2-α- d -glucopyranosyl-stachyose, 2 G (2-α- d -glucopyranosyl) 2 -stachyose and 2 G (2-α- d -glucopyranosyl) 3 -stachyose.
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synthesis and structural analysis of five novel oligosaccharides prepared by glucosyltransfer from β d Glucose 1 Phosphate to isokestose and nystose using thermoanaerobacter brockii kojibiose phosphorylase
Carbohydrate Research, 2003Co-Authors: Hideki Okada, Tomoyuki Nishimoto, Eri Fukushi, Shuichi Onodera, Jun Kawabata, Masanori Kikuchi, Norio ShiomiAbstract:Abstract Five novel oligosaccharides (tetra-, penta- and hexa-saccharides) were synthesized by glucosyltransfer from β- d -Glucose 1-Phosphate to isokestose ( O -β- d -fructofuranosyl-(2→1)- O -β- d -fructofuranosyl-(2→1)-α- d -glucopyranoside) or nystose ( O -β- d -fructofuranosyl-(2→1)- O -β- d -fructofuranosyl-(2→1)- O -β- d -fructofuranosyl-(2→1)-α- d -glucopyranoside) using Thermoanaerobacter brockii kojibiose phosphorylase. The oligosaccharides were identified as 2(2-α- d -glucopyranosyl) m isokestose; [ O -α- d -glucopyranosyl-(1→2)] m - O -[β- d -fructofuranosyl-(2→1)] 2 -α- d -glucopyranoside: m =1, 2, and 3, and 2(2-α- d -glucopyranosyl) n nystose; [ O -α- d -glucopyranosyl-(1→2)] n - O -[β- d -fructofuranosyl-(2→1)] 3 -α- d -glucopyranoside: n =1 and 2 using gas liquid chromatography analysis of the methyl derivatives, and MALDI-TOF-MS and NMR measurements of the newly formed oligosaccharides. 1 H, 13 C NMR signals of each saccharide were assigned using 2D-NMR techniques, including COSY, HSQC, HSQC–TOCSY, HMBC, CH 2 -selected E-HSQC, and CH 2 -selected E-HSQC–TOCSY.
Jack Preiss - One of the best experts on this subject based on the ideXlab platform.
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molecular architecture of the Glucose 1 Phosphate site in adp Glucose pyrophosphorylases
Journal of Biological Chemistry, 2006Co-Authors: Clarisa M Bejar, Miguel A Ballicora, Xiangshu Jin, Jack PreissAbstract:ADP-Glc pyrophosphorylase (PPase), a key regulatory enzyme in the biosynthetic pathway of starch and bacterial glycogen, catalyzes the synthesis of ADP-Glc from Glc-1-P and ATP. A homology model of the three-dimensional structure of the Escherichia coli enzyme complexed with ADP-Glc has been generated to study the substrate-binding site in detail. A set of amino acids in the model has been identified to be in close proximity to the Glucose moiety of the ADP-Glc ligand. The role of these amino acids (Glu194, Ser212, Tyr216, Asp239, Phe240, Trp274, and Asp276) was studied by site-directed mutagenesis through the characterization of the kinetic properties and thermal stability of the designed mutants. All purified alanine mutants had 1 or 2 orders of magnitude lower apparent affinity for Glc-1-P compared with the wild type, indicating that the selected set of amino acids plays an important role in their interaction with the substrate. These amino acids, which are conserved within the ADP-Glc PPase family, were replaced with other residues to investigate the effect of size, hydrophobicity, polarity, aromaticity, or charge on the affinity for Glc-1-P. In this study, the architecture of the Glc-1-P-binding site is characterized. The model overlaps with the Glc-1-P site of other PPases such as Pseudomonas aeruginosa dTDP-Glc PPase and Salmonella typhi CDP-Glc PPase. Therefore, the data reported here may have implications for other members of the nucleotide-diphosphoGlucose PPase family.
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mutagenesis of the Glucose 1 Phosphate binding site of potato tuber adp Glucose pyrophosphorylase
Plant Physiology, 1998Co-Authors: Miguel A Ballicora, Jack PreissAbstract:Lysine (Lys)-195 in the homotetrameric ADP-Glucose pyrophosphorylase (ADPGlc PPase) from Escherichia coli was shown previously to be involved in the binding of the substrate Glucose-1-Phosphate (Glc-1-P). This residue is highly conserved in the ADPGlc PPase family. Site-directed mutagenesis was used to investigate the function of this conserved Lys residue in the large and small subunits of the heterotetrameric potato ( Solanum tuberosum ) tuber enzyme. The apparent affinity for Glc-1-P of the wild-type enzyme decreased 135- to 550-fold by changing Lys-198 of the small subunit to arginine, alanine, or glutamic acid, suggesting that both the charge and the size of this residue influence Glc-1-P binding. These mutations had little effect on the kinetic constants for the other substrates (ATP and Mg 2+ or ADP-Glc and inorganic Phosphate), activator (3-phosphoglycerate), inhibitor (inorganic Phosphate), or on the thermal stability. Mutagenesis of the corresponding Lys (Lys-213) in the large subunit had no effect on the apparent affinity for Glc-1-P by substitution with arginine, alanine, or glutamic acid. A double mutant, S K198R L K213R , was also obtained that had a 100-fold reduction of the apparent affinity for Glc-1-P. The data indicate that Lys-198 in the small subunit is directly involved in the binding of Glc-1-P, whereas they appear to exclude a direct role of Lys-213 in the large subunit in the interaction with this substrate.