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• W2017189404 abstract "Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel β-helix (LβH), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors. Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel β-helix (LβH), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors. Uridinediphosphate-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) 2The abbreviations used are: UGPase, UDP-glucose pyrophosphorylase; LβH, left-handed parallel β-helix; UDP-glucose, uridine diphosphate-glucose; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; r.m.s., root mean square; NB, nucleotide binding; SB, substrate binding. 2The abbreviations used are: UGPase, UDP-glucose pyrophosphorylase; LβH, left-handed parallel β-helix; UDP-glucose, uridine diphosphate-glucose; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; r.m.s., root mean square; NB, nucleotide binding; SB, substrate binding. is present in all three kingdoms of life and catalyzes the reaction of UTP + glucose 1-phosphate → UDP-glucose + PPi in the presence of Mg2+ in vivo. UDP-glucose, the activated form of glucose, plays an essential role in the metabolism of carbohydrates in all organisms. UDP-glucose is the main glucosyl donor for the formation of glycogen, starch, and cellulose, as well as for the synthesis of glucose-containing glycolipids, glycoproteins, and proteoglycans (1Flores-Diaz M. Alape-Giron A. Persson B. Pollesello P. Moos M. von Eichel-Streiber C. Thelestam M. Florin I. J. Biol. Chem. 1997; 272: 23784-23791Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 2Ordin L. Hall M.A. Plant Physiol. 1968; 43: 473-476Crossref PubMed Scopus (43) Google Scholar). In addition, other important nucleotide sugars such as UDP-xylose, UDP-glucuronic acid, and UDP-galactose are derived from UDP-glucose. In bacteria some of these activated sugars are used to build the bacterial polysaccharide capsule that often represents the sole determinant of virulence of these organisms. In Streptococcus pneumoniae, for example, it was known that mutants containing an inactivated UGPase gene (galU) are completely avirulent, as they are unable to form the polysaccharide capsule (3Bonofiglio L. Garcia E. Mollerach M. Curr. Microbiol. 2005; 51: 217-221Crossref PubMed Scopus (49) Google Scholar). Similarly, UGPase is involved in the biosynthesis of the lipopolysaccharide core in Escherichia coli, resulting in a reduced adhesion behavior of E. coli galU mutants (4Genevaux P. Bauda P. DuBow M.S. Oudega B. Arch. Microbiol. 1999; 172: 1-8Crossref PubMed Scopus (113) Google Scholar). The protozoan parasite Leishmania is the causative agent of a widespread group of diseases collectively known as Leishmaniasis. The disease affects more than 12 million people world-wide and until now there is no specific drug available to cure the disease. 3World Health Organization (special program for research and training in tropical diseases). 3World Health Organization (special program for research and training in tropical diseases). Leishmania express various glycoconjugates on their cell surface that is dynamically modified during the parasite life cycle allowing the survival and proliferation in the sand fly vector as well as in the mammalian host (6Naderer T. Vince J.E. McConville M.J. Curr. Mol. Med. 2004; 4: 649-665Crossref PubMed Scopus (115) Google Scholar, 7Sacks D.L. Modi G. Rowton E. Spath G. Epstein L. Turco S.J. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 406-411Crossref PubMed Scopus (171) Google Scholar). The biosynthesis of glycoconjugates essentially depends on the availability of activated nucleotide sugars. UGPase represents a key position in the activation of glucose and galactose, which are major components of Leishmania glycoconjugates. Formation of UDP-glucose is a prerequisite for the synthesis of UDP-galactose, which is achieved either by nucleotide transfer from UDP-glucose onto galactose 1-phosphate or by epimerization of UDP-glucose to UDP-galactose. As galactose-containing glycoconjugates are important virulence factors in Leishmania major, its UGPase has been intensively characterized. Like other pyrophosphorylases the L. major UGPase requires a divalent metal ion, Mg2+, and acts reversibly in vitro. The enzymatic reaction follows an ordered bi-bi reaction mechanism with sequential binding of UTP preceding glucose 1-phosphate. Interestingly, UDP and UMP are not recognized by the enzyme and do not facilitate the binding site for glucose 1-phosphate (8Lamerz A.-C. Haselhorst T. Bergfeld A.K. von Itzstein M. Gerardy-Schahn R. J. Biol. Chem. 2006; 281: 16314-16322Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Thus, the γ-phosphate group of UTP is essential for both binding of the nucleotide and inducing of the presumed change in conformation, which then allows the glucose 1-phosphate to the active site. Several mutagenesis studies and a modeling approach in eukaryotic UGPases have been performed, which identified specific residues important for catalytic activity (9Katsube T. Kazuta Y. Tanizawa K. Fukui T. Biochemistry. 1991; 30: 8546-8551Crossref PubMed Scopus (47) Google Scholar, 10Chang H.Y. Peng H.L. Chao Y.C. Duggleby R.G. Eur. J. Biochem. 1996; 236: 723-728Crossref PubMed Scopus (16) Google Scholar, 11Martz F. Wilczynska M. Kleczkowski L.A. Biochem. J. 2002; 367: 295-300Crossref PubMed Scopus (57) Google Scholar, 12Geisler M. Wilczynska M. Karpinski S. Kleczkowski L.A. Plant Mol. Biol. 2004; 56: 783-794Crossref PubMed Scopus (33) Google Scholar). However, these investigations alone were not enough to resolve the catalytic mechanism. The knowledge of the three-dimensional structure is, therefore, of utmost importance. In this study we succeeded in solving the three-dimensional structure of the L. major UGPase apoprotein and of the complex of UGPase with the product UDP-glucose. Comparison of the two structures revealed an induction from an open to a closed conformation when the product is bound accompanied by major domain motions. In combination with mutagenesis data, our results provide first insight into the mechanism of UDP-glucose formation and establish the structural basis for the rational design of inhibitors that specifically affect the L. major enzyme. Protein Expression and Purification—The gene encoding UGPase from L. major was subcloned into the His tag expression vector pET-22b (Novagen). The recombinant plasmid pET-UGP-His was transformed into E. coli expression strain BL21(DE3), overexpressed, and purified by Ni2+ chelating chromatography as described previously (8Lamerz A.-C. Haselhorst T. Bergfeld A.K. von Itzstein M. Gerardy-Schahn R. J. Biol. Chem. 2006; 281: 16314-16322Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). As a last additional purification step the buffer of the purified protein was changed by using a HiTrap desalting column (Amersham Biosciences) to buffer A (1 mm dithiothreitol, 2 mm MgCl2, 10 mm Tris-HCl, pH 7.5, 100 mm NaCl). Finally, the protein was concentrated to 20 mg/ml. Selenomethionine-labeled protein was produced in the methionine-auxothrophic E. coli strain B834(DE3) and grown in minimal medium without methionine supplemented with selenomethionine (0.3 mm). After induction with 1 mm isopropyl 1-thio-β-d-galactopyranoside the cells were grown at 15 °C for 18 h. The purification procedure was identical to that of the native protein. Incorporation of selenomethionine was confirmed by determination of the molecular mass using ESI-MS. Site-directed Mutagenesis—Plasmids for the expression of mutant UGPase were generated by site-directed mutagenesis of pET-UGP-His using the QuikChange Site-directed Mutagenesis kit (Stratagene) according to the manufacturer's instructions. The following mutagenic primers were used: 5′-GGGCTGTGCGACGCCGCGACGCTGCTCGAGGTC-3′ (K95A); 5′-GGGCGCCGCCGGGGAACGGTGACATCTAC-3′ (H191N); 5′-GGGCGCCGCCGGGGCTCGGTGACATCTAC-3′ (H191L); 5′-GAGAAGAGGGTGCTGGATCTGCGGGAGTCCGCC-3′ (L281D); and 5′-CTTCGCGCCAGTGGCGACGTGCGCCGATC-3′ (K380A). Integrity of the constructs was confirmed by sequencing. The mutants were expressed and purified as described above. In Vitro Activity Measurement—The in vitro activity of mutant and wild type UGPase was measured in the forward reaction by a coupled enzymatic assay as described previously (8Lamerz A.-C. Haselhorst T. Bergfeld A.K. von Itzstein M. Gerardy-Schahn R. J. Biol. Chem. 2006; 281: 16314-16322Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Briefly, the assay was performed at 25 °C in 50 mm Tris-HCl, pH 7.8, 10 mm MgCl2, 1 mm 2-mercaptoethanol, 1 mm UTP (Roche), 1 mm glucose 1-phosphate (Sigma), 1 mm NAD+ (Roche Applied Science), and 0.05 units of UDP-glucose dehydrogenase (Calbiochem). The reaction was started by the addition of wild type or mutant UGPase. The reduction of the cofactor NAD+ to NADH + H+ was monitored at 340 nm and the initial linear rates were used to calculate the enzymatic activity. Crystallization and Data Collection—Crystals were grown at 18 °C using the sitting drop vapor diffusion method. As the crystallization of selenomethionine-labeled protein yielded better diffracting crystals, the labeled protein was used for all trials. Crystals of the apoprotein were grown from 21% PEG 3350, 100 mm BisTris, pH 7.0, 200 mm Li2SO4. Crystals of UGPase in complex with UDP-glucose were obtained from 27% PEG monomethyl ether 2000, 100 mm BisTris, pH 6.5. For co-crystallization UDP-glucose (5 mm) was used in excess. All crystals belong to space group C2221 and contain one molecule per asymmetric unit. Diffraction data of the apoprotein as well as the multiwavelength anomalous dispersion data sets of the complex were collected at beamline BW6 at the German Electron Synchrotron (Hamburg, Germany). Data sets were integrated, scaled, and merged by using DENZO and SCALEPACK (13Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38436) Google Scholar). The data collection statistics are summarized in Table 1.TABLE 1Data collection and phasing statisticsData setsUDP-glucoseApoPeakInflectionRemoteWavelength (Å)0.979200.979600.9501.050Space GroupC2221C2221Unit cell (Å)a80.273.0b89.9107.7c137.2150.1α = β = γ90.090.0Resolution (Å)20-2.33 (2.35-2.33)20-2.4 (2.43-2.40)Unique reflections21,22621,02621,00121,635Redundancy7.03.63.63.1Completeness (%)98989692.1Atomic completeness (%)989796I/σ(I)67 (42)46 (28)46 (28)19 (2.3)Rmerge0.06 (0.1)0.06 (0.1)0.06 (0.09)0.06 (0.45)Mean figure of merit (FOM) before DM0.73Mean figure of merit (FOM) after DM0.80 Open table in a new tab Phase Determination and Structure Refinement—The structure of the UGPase + UDP-glucose complex was solved by the multiwavelength anomalous dispersion phasing method with programs from the CCP4 collection (14Collaborative Computational Project 4Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19728) Google Scholar). The positions of 16 selenomethionine atoms present in the asymmetric unit of the complexed form were determined using the program SHELXD (15Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-343Crossref PubMed Scopus (1877) Google Scholar). Data were phased and refined to a resolution of 2.3 Å with MLPHARE. Initial phases were improved by solvent flattening with the program DM from the CCP4 package (Table 1). The resulting electron density map was used in an automated structure refinement approach. ARP/wARP including the warpNtrace protocol (16Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2563) Google Scholar) were used to obtain a first glycine trace of the protein structure. Starting from this initial model the complete catalytic domain and most of the N-terminal domain of the model could be built. The model was refined in CNS (17Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16946) Google Scholar) and rebuilt in O (18Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar). The loop region between the catalytic domain and the C-terminal domain was not well defined in the electron density of the ligand bound structure, whereas this part was traceable in the apo structure. The apo structure was solved by molecular replacement using the likelihood-based molecular replacement program PHASER-1.2 (19McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1596) Google Scholar). The coordinates of 482 residues could be refined using the excellent difference electron density map, except for the first six and the last six residues. The loop containing residues 267 to 274 and residues 467 to 469 showed weak density. It was not possible to detect any electron density in the two structures, which could be assigned to Mg2+. However, in the apo structure two peaks of electron density are located at the active site and at the protein surface, which apparently are sulfate ions (SO2-4) from the screening buffer. The refinement was done with CNS and the model rebuilding was performed in O. The well defined C-terminal domain of the apo structure was used to solve the missing C-terminal domain of the ligand structure by molecular replacement. The final model of the ligand structure contained all residues beside the first and the last six residues of the full-length protein. The ligand UDP-glucose was built into the model with the automated ligand building module from ARP/wARP version 6.1. Averaged Sigma-A-weighted and composite omit (2Fo - Fc) maps were used for model building of the ligand in O. Water molecules were added and refined by CNS. The statistical data of the refinement are presented in Table 2, including the Protein Data Bank accession codes.TABLE 2Refinement statisticsData setsUDP-glucoseApoProtein atoms in the asymmetric unit37223722Solvent content (%)41.446.0Solvent atoms22575SC42− ions2LigandUDP-glucoseResolution (Å)20-2.320-2.4RcrysaRcrys = ∑|Fobs - Fcalc|/∑Fobs.0.21 (0.25)0.21 (0.30)RfreebRfree was determined from 5% of the data (10% of the apo dataset) that were omitted from the refinement.0.25 (0.31)0.26 (0.35)R.m.s.deviation bond lengths (Å)0.0080.006R.m.s.deviation bond angles (°)1.51.3Protein B-factor (Å2)28.048.7Ligand B-factor (Å2)17.0Ramachandran φ/ψ distribution (%)cRamachandran plot distribution refers to the most favored/additional/generously/disallowed regions as defined by Procheck (5).90.2/9.1/0/0.787.2/11.8/0.7/0.2PDB entry2OEG2OEFa Rcrys = ∑|Fobs - Fcalc|/∑Fobs.b Rfree was determined from 5% of the data (10% of the apo dataset) that were omitted from the refinement.c Ramachandran plot distribution refers to the most favored/additional/generously/disallowed regions as defined by Procheck (5Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Open table in a new tab Searches for related sequences were performed with BLAST, searches for related structures were done with DALI. Sequence and structural alignments were done with GCG (Wisconsin Package version 10.3, Accelrys, San Diego, CA) and TOP3D (20Lu G. Protein Data Bank Quarterly Newsletter. 1996; 78: 10-11Google Scholar), respectively. Calculation of the tilt angles of the domain motion was done with DynDom. Figures were generated with PYMOL, MOLSCRIPT (21Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), RASTER3D (22Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3873) Google Scholar), and ALSCRIPT (23Barton G.J. Protein Eng. 1993; 6: 37-40Crossref PubMed Scopus (1110) Google Scholar). The 2.3-Å resolution structure of the 54.5-kDa full-length L. major UGPase complexed with UDP-glucose (closed conformation) was solved by multiwavelength anomalous dispersion using selenomethionine-substituted protein. Except for a small part of the N and C terminus the whole model (S7-P488) could be built with two surface loops (Lys267-Asp274 and Asn466-Ser471) associated with weak electron density. The model has overall good stereochemistry with 99% of the residues in the preferred Φ/ψ conformation and final R values of Rcrys = 0.21 and Rfree = 0.25. Also, the substrate-free apoprotein (open conformation) crystallized in the space group C2221 but with a different unit cell and its structure was solved by molecular replacement. The model of the apoprotein was refined to 2.4 Å with final R values of Rcrys = 0.21 and Rfree = 0.26. In the apo model 87% of the residues displayed a main chain conformation in the most favored regions and 12% in the additional allowed regions as defined by the Ramachandran plot. All residues with unusual main chain conformation were either very well defined in the electron density map or occupied flexible regions of the model. A summary of the data collection is presented in Table 1 and the refinement statistics are summarized in Table 2. Overall Structure—UGPase contains an N-terminal domain, a central catalytic domain, and a C-terminal domain (Fig. 1, A and B). The catalytic domain harbors the nucleotide sugar binding site and resembles the Rossmann-fold seen in many nucleotide-binding proteins. It consists of a central highly bent, twisted and mixed sheet composed of seven β-strands arranged in the order 4-3-1-7-11-8-14 that are parallel aligned except β11. This central sheet is topped at one end by a small two-stranded anti-parallel β-sheet (β2a-β2b) and flanked at the opposite side by a long two-stranded anti-parallel β-sheet (β9-β10), which points to a protruding extension. The central sheet is additionally surrounded by 11 α-helices and seven 310 helices. The side of the central sheet that faces the N-terminal domain is partially covered by the α-helix α8. In contrast, the top side of the central sheet is solvent accessible and faces the nucleotide sugar moiety. Furthermore, a sequence insertion in the Leishmania enzyme forms the long loop (residues Arg261-Leu280) protruding from the catalytic domain, which has the appearance of a handle (Fig. 1B). The N-terminal domain is discontinuously formed by residues Ser7-Thr44 encompassing the two N-terminal α-helices α1 and α2, and two further building blocks (residues Asn163-Pro188 and Pro331-Ala355) that protrude from the catalytic domain (Fig. 1B). These segments contain a four-stranded anti-parallel β-sheet (β5a, β6, β12, and β13), flanked on one side by α1 and α2 and on the other side by the short β-strand β5b and a310 helix. The C-terminal domain is connected to the catalytic domain via a long loop that encompasses two anti-parallel β-strands (β15 and β16) and a 310 helix. The domain comprises residues Asp391-Pro488 that are arranged in a left-handed parallel β-helix (LβH) composed of three complete rungs. The first rung contains a large loop and is atypically composed of a shortened β-strand, two subsequent α-helices, and another β-strand. The second and the third rung are formed by three canonical short β-strands. The LβH is assembled by tandem repeats of imperfect hexa-peptides, which harbor strongly conserved hydrophobic residues at the so-called i-positions (Fig. 1B) pointing toward the interior of the LβH. Generally, small residues occupy the corner positions at the sharp turns. Active Site—The refined structure of L. major UGPase in complex with UDP-glucose, the product of the enzymatic reaction, defines the location of the active site of the enzyme (Fig. 2A). Fig. 2B shows the well defined electron density corresponding to UDP-glucose. The UDP-glucose is bound in a deep groove located at the center of the catalytic domain that consists of highly conserved residues. The nucleotide moiety is fixed by a mixture of hydrogen bonds and hydrophobic interactions established by residues of the conserved NB loop (nucleotide binding loop (Fig. 1B)). The NB loop (residues Lys80-Lys95), which constitutes a roof above the nucleotide on the active site, contains the glycine-rich consensus sequence motif KLNGGLGTXMG(X)4K. The basic residues His191 and Lys380 interact with the negatively charged phosphate groups of UDP-glucose (Fig. 3A). The glucose moiety of the product is bound to a depression that is adjacent to the nucleotide. The hydroxyl groups of the sugar are fixed by numerous hydrogen bonds (Fig. 3A). These observations are in good agreement with data obtained from saturation transfer difference-NMR, indicating that all protons of the sugar moiety are in intimate contact with the protein (8Lamerz A.-C. Haselhorst T. Bergfeld A.K. von Itzstein M. Gerardy-Schahn R. J. Biol. Chem. 2006; 281: 16314-16322Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). At the end of the cleft Phe305 contacts the sugar moiety in the closed conformation, thereby limiting the space available for sugar binding. The SB loop (substrate binding loop, residues Thr250-Gly257) covers the sugar within the binding site and contains the two highly conserved glycines, Gly256 and Gly257, that rearrange during the catalytic reaction (see below).FIGURE 3A, close up view of the active site. Important residues of the closed conformation are shown as blue sticks with blue labels. Residues of the open conformation are displayed as magenta lines and important glycines of the NB and SB loops as well as Lys380 and Phe305 of the open conformation are labeled in magenta. UDP-glucose is depicted in cyan. Hydrogen bonds with the product are marked by dotted green lines. B, identical view on the active site. The modeled UTP and glucose 1-phosphate are shown in cyan. Their phosphorus atoms are depicted in gray, whereas the sulfate ion is given in yellow. Important residues of the open conformation probably involved in substrate binding are shown in magenta.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The binding mode of UDP-glucose and the active site architecture of UGPase in general provide information about a putative binding mechanism of the UGPase substrate UTP. When the nucleotide moiety of UTP is superimposed with the uridyl group of UDP-glucose, the β- and γ-phosphate groups of UTP can be positioned into a small cavity above the nucleotide binding site that is lined by Lys380 and Lys95 (Fig. 3B) leading to a conformation of UTP similar to that of dTTP in the structurally related glucose-1-phosphate thymidylyltransferase (RmlA/RffH) (24Blankenfeldt W. Asuncion M. Lam J.S. Naismith J.H. EMBO J. 2000; 19: 6652-6663Crossref PubMed Scopus (161) Google Scholar, 25Sivaraman J. Sauve V. Matte A. Cygler M. J. Biol. Chem. 2002; 277: 44214-44219Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Based on our structural data we mutated several substrate binding residues in the active site of UGPase to further study the importance of these residues for catalysis. Lys380 forms a hydrogen bond to the α-phosphate group of UDP-glucose. Most probably, both residues Lys380 and Lys95 bind to the phosphate groups of the substrate UTP. Therefore, no catalytic activity for the K380A mutant was observed, in contrast to a residual activity of 0.5% for the K95A mutant (Table 3). Residue His191 binds and stabilizes the β-phosphate group of UDP-glucose and probably also the phosphate group of the substrate glucose 1-phosphate. When His191 is substituted by the apolar leucine, a residue of similar size, the enzyme is inactive because the hydrogen bond between histidine and the β-phosphate is abolished. In the H191N mutant asparagine may still be able to form a hydrogen bond to the substrate, which is in agreement with the observed reduced activity of 0.3% of the H191N mutant and the natural occurrence of asparagine at position 223 in the human UDP-N-acetylglucosamine PPase (AGX) (26Peneff C. Ferrari P. Charrier V. Taburet Y. Monnier C. Zamboni V. Winter J. Harnois M. Fassy F. Bourne Y. EMBO J. 2001; 20: 6191-6202Crossref PubMed Scopus (120) Google Scholar).TABLE 3In vitro activity of wild-type and mutant enzymesEnzyme VariantSpecific activityaData are the mean ± S.D from at least three independently performed assays.Specific activityRef.units/mg%Wild type1477 ± 157100This workK95A7.8 ± 1.40.5This workH191N3.7 ± 0.10.3This workH191L0.00.0This workL281D240 ± 2816.3This workK380A0.00.0This workG84D4% residual activity1K380QInactive9a Data are the mean ± S.D from at least three independently performed assays. Open table in a new tab Comparison of Open and Closed Forms—Upon product binding and presumably also upon substrate binding UGPase undergoes a large conformational change that involves a rearrangement of the C-terminal domain toward the active site and in functional loops of the catalytic domain. Superposition of the catalytic domain in the open and closed conformation results in a r.m.s. deviation of 3.4 Å in the 301 position of Cα-atoms. In comparison the N- and C-terminal domains can be superimposed with r.m.s. deviation values of 0.3 and 0.7 Å, respectively. The latter domain of the open form is tilted toward the catalytic domain about 17° in the closed conformation (Figs. 2A and 4). The superposition of both structures in the open and the closed conformations reveal a significant relocation of the NB loop toward the ligand (Fig. 2A). On the opposite edge of the active site the SB loop with the conserved glycines, Gly256 and Gly257, moves over the sugar moiety of the product and the side chain of Phe305 rearranges to tighten up the active site (Figs. 2A, 3A, and 4). The handle-like extension formed by β-strands, β9 and β10, and the connecting loop adopts very different conformations in the apo and the UDP-glucose-complexed UGPase structures. Currently, the role of the handle movement is not clear. However, residues Gly256 and Gly257 at the beginning and Glu284 at the end of the handle, as well as the adjacent residues Val246 to Ala260 and Leu281 to Ser303 perform a 12° turn toward the sugar moiety in the UDP-glucose UGPase complex compared with the apo structure (Figs. 2A and 4). This fact justifies the assumption that the large movement of the handle is at least partially induced by ligand binding. UGPases of eukaryotic origin are about 500 residues long and show 40% similarity in protein sequence (Fig. 1B). Prokaryotic UGPases in general are shorter and their sequences align with a very limited degree of conservation. Nevertheless, both human as well as yeast UGPase are able to complement UGPase (GalU)-deficient E. coli mutants (27Peng H.L. Chang H.Y. FEBS Lett. 1993; 329: 153-158Crossref PubMed Scopus (31) Google Scholar, 28Daran J.M. Dallies N. Thines-Sempoux D. Paquet V. Francois J. Eur. J. Biochem. 1995; 233: 520-530Crossref PubMed Scopus (106) Google Scholar). In contrast to the monomeric L. major and plant UGPases (8Lamerz A.-C. Haselhorst T. Bergfeld A.K. von Itzstein M. Gerardy-Schahn R. J. Biol. Chem. 2006; 281: 16314-16322Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 11Martz F. Wilczynska M. Kleczkowski L.A. Biochem. J. 2002; 367: 295-300Crossref PubMed Scopus (57) Google Scholar) the active animal and fungal UGPases are oligomeric. The enzyme, Ugp1p from Saccharomyces cerevisiae forms octamers in" @default.
• W2017189404 created "2016-06-24" @default.
• W2017189404 creator A5001569569 @default.
• W2017189404 creator A5026167637 @default.
• W2017189404 creator A5032737644 @default.
• W2017189404 creator A5034781403 @default.
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• W2017189404 date "2007-04-01" @default.
• W2017189404 modified "2023-09-30" @default.
• W2017189404 title "Open and Closed Structures of the UDP-glucose Pyrophosphorylase from Leishmania major" @default.
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