Kinetic and crystallographic analyses support a sequential-ordered bi bi catalytic mechanism for Escherichia coli glucose-1-phosphate thymidylyltransferase

Zuccotti, Simone; Zanardi, Davide; Rosano, Camillo; Sturla, Laura; Tonetti, Michela; Bolognesi, Martino

doi:10.1006/jmbi.2001.5073

Cited by 98 publications

(120 citation statements)

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“…It should be noted that, in the thymidylyltransferase/ product complex models presently available, there are no bound magnesium ions observed in the active sites (Blankenfeldt et al 2000;Zuccotti et al 2001). Contrastingly, in the structure of the thymidylyltransferase solved in the presence of dTTP, there is a single magnesium ion bound to the nucleotide and the carboxylate groups of Asp 108 and Asp 223 (Sivaraman et al 2002).…”

Section: Resultsmentioning

confidence: 96%

“…There is a total 13 b-strands that form three layers of sheet. The first and largest layer is a mixed b-sheet consisting of b-strands 1, 2,3,4,6,8,11,12 A search with DALI (Holm and Sander 1996) reveals that the closest structural relatives to UGPase are the glucose-1-phosphate thymidylyltransferases (Blankenfeldt et al 2000;Barton et al 2001;Zuccotti et al 2001;Sivaraman et al 2002) and the bifunctional N-acetylglucosamine-1-phosphate uridylyltransferase (Brown et al 1999). A superposition of the UGPase subunit (304 residues) onto that of the E. coli thymidylytransferase (293 residues) is presented in Figure 4A.…”

Section: Resultsmentioning

confidence: 99%

“…From this study the tertiary and quaternary structures of the enzyme have been defined. As described, UGPase adopts a molecular fold similar to that of glucose-1-phosphate thymidylyltransferase (Blankenfeldt et al 2000;Barton et al 2001;Zuccotti et al 2001;Sivaraman et al 2002) and UDP-N-acetylglucosamine pyrophosphorylase (Brown et al 1999). These two bacterial enzymes play key roles in the synthesis of activated sugars, which ultimately serve as precursors for the synthesis of cell-surface structures.…”

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confidence: 92%

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The molecular architecture of glucose‐1‐phosphate uridylyltransferase

Thoden

Holden

2007

Protein Science

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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 Å resolution 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-and 39-hydroxyl groups of the glucosyl moiety. Details concerning the overall structure of UGPase and a comparison with glucose-1-phosphate thymidylyltransferase are presented.Keywords: UDP-glucose; glucose-1-phosphate uridylyltransferase; UDP-glucose pyrophosphorylase; X-ray structure; carbohydrates The conversion of b-D-galactose to the more metabolically useful glucose-1-phosphate is accomplished in most organisms by the action of four enzymes that constitute the Leloir pathway (Holden et al. 2003). In the first step of this pathway, b-D-galactose is epimerized to a-D-galactose by galactose mutarotase. The next step involves the ATP-dependent phosphorylation of a-Dgalactose by galactokinase to yield galactose-1-phosphate. Subsequently, in the third step, the enzyme galactose-1-phosphate uridylyltransferase catalyzes the transfer of a UMP group from UDP-glucose to galactose-1-phosphate, thereby generating glucose-1-phosphate and UDP-galactose. To complete the pathway, UDP-galactose is converted to UDP-glucose by UDPgalactose 4-epimerase. In humans, mutations in the genes that encode the galactokinase, the galactose-1-phosphate uridylyltransferase, or the epimerase can result in the diseased state referred to as galactosemia with clinical manifestations including intellectual retardation, speech disorders, liver dysfunction, and cataract formation. The most common form of galactosemia arises from defects in galactose-1-phosphate uridylyltransferase and as such it has been the subject of intensive kinetic and structural investigations for many years (Holden et al. 2003). Studies have...

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

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confidence: 92%

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The molecular architecture of glucose‐1‐phosphate uridylyltransferase

Thoden

Holden

2007

Protein Science

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“…11 Nucleotidylyltransferases are an attractive antimicrobial target, in that they show broad homology across various species. 12 Cps2L (EC 2.7.7.24) is a thymidylyltransferase responsible for coupling α-D-glucose 1-phosphate (1, Glc-1-P) and deoxythymidine triphosphate (2, dTTP) to form deoxythymidine diphosphate-α-D-glucose (3, dTDP-Glc) ( Figure 1). RmlB (EC 4.2.1.46) is responsible for oxidation of the C4″ hydroxyl unit and C6″ dehydration.…”

Section: ■ Introductionmentioning

confidence: 99%

Synthesis and Evaluation of l-Rhamnose 1C-Phosphonates as Nucleotidylyltransferase Inhibitors

et al. 2013

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/npsi/ctrl?lang=en http://nparc.cisti-icist.nrc-cnrc.gc.ca/npsi/ctrl?lang=fr Access and use of this website and the material on it are subject to the Terms and Conditions set forth at http://nparc.cisti-icist.nrc-cnrc.gc.ca/npsi/jsp/nparc_cp.jsp?lang=en NRC Publications Archive Archives des publications du CNRCThis publication could be one of several versions: author's original, accepted manuscript or the publisher's version. / La version de cette publication peut être l'une des suivantes : la version prépublication de l'auteur, la version acceptée du manuscrit ou la version de l'éditeur. For the publisher's version, please access the DOI link below./ Pour consulter la version de l'éditeur, utilisez le lien DOI ci-dessous.http://dx.doi.org/10.1021/jo401542sThe Journal of Organic Chemistry, 78, 19, pp. 9822-9833, 2013-09-10 ABSTRACT: We report the synthesis of a series of phosphonates and ketosephosphonates possessing an L-rhamnose scaffold with varying degrees of fluorination. These compounds were evaluated as potential inhibitors of α-D-glucose 1-phosphate thymidylyltransferase (Cps2L), the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis, and a novel antibiotic target. Enzyme−substrate and enzyme−inhibitor binding experiments were performed using water-ligand observed binding via gradient spectroscopy (WaterLOGSY) NMR for known sugar nucleotide substrates and selected phosphonate analogues. IC 50 values were measured and K i values were calculated for inhibitors. New insights were gained into the binding promiscuity of enzymes within the prokaryotic L-rhamnose biosynthetic pathway (Cps2L, RmlB−D) and into the mechanism of inhibition for the most potent inhibitor in the series, L-rhamnose 1C-phosphonate. ■ INTRODUCTIONStreptococcus pneumoniae is a prevalent, highly infectious, Gram-positive pathogen that has shown high clinical resistance to penicillin and chloramphenicol. 1 The mechanisms of resistance for S. pneumoniae and other more invasive pathogens, including Mycobacterium tuberculosis, usually entail alterations to drug target proteins involved in cell wall synthesis. 2−5 Bacterial chemo-resistance, including resistance to synergistic treatments using β-lactams and aminoglycosides, is a growing barrier to the treatment of invasive pathogens (i.e., S. pneumoniae and M. tuberculosis). 6 Cell wall biosynthesis is a commonly pursued target for a variety of bacterial enzyme inhibitors as cell wall assembly is essential for bacterial survival and virulence. L-Rhamnose (6-deoxy-L-mannose) serves as the linking unit between arabinogalactan and peptidoglycan 7,8 and is a necessary constituent of the bacterial cell wall in many bacterial species. 9 The biosynthesis of L-rhamnose begins with nucleotidylyltransferase enzymes (Cps2L/RmlA) responsible for generating activated sugars in the form of glycosylated nucleoside diphosphates (NDPs). These NDPs are generated from the enzymatic coupling of sugar 1-phosphates with nucleoside triphosphates (NTPs). Once formed, the NDPs may be further modified ...

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“…Periplasmic glucan biosynthesis protein OpgH is a glucosyltransferase that catalyzes the elongation of beta-1,2 polyglucose chains of glucan, requiring a beta-glucoside as a primer and UDP-glucose as a substrate [27]. The central cytoplasmic region of OpgH shares strong structural identity with glucosyl-transferases in which several aspartic acid residues are needed for its catalytic activity [17,28,29].…”

Section: Requirement Of Full Length Opggh Protein For Swarm Motilitymentioning

confidence: 99%

Swarm and swim motilities of Salmonella enterica serovar Typhimurium and role of osmoregulated periplasmic glucans

Dharne¹,

Porteen²,

Murphy³

et al. 2015

Microbiol Discov

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Kinetic and crystallographic analyses support a sequential-ordered bi bi catalytic mechanism for Escherichia coli glucose-1-phosphate thymidylyltransferase

Cited by 98 publications

References 45 publications

The molecular architecture of glucose‐1‐phosphate uridylyltransferase

The molecular architecture of glucose‐1‐phosphate uridylyltransferase

Synthesis and Evaluation of l-Rhamnose 1C-Phosphonates as Nucleotidylyltransferase Inhibitors

Swarm and swim motilities of Salmonella enterica serovar Typhimurium and role of osmoregulated periplasmic glucans

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