Exploiting Nucleotidylyltransferases To Prepare Sugar Nucleotides

Timmons, Shannon C.; Mosher, Roy H.; Knowles, Sheryl A.; Jakeman, David L.

doi:10.1021/ol0630853

Cited by 49 publications

(82 citation statements)

References 35 publications

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“…PEPs was overexpressed and purified using standard methods for His 6 -tagged proteins as previously described (Timmons et al 2007;Jakeman et al 2008) with the following modifications. Protein overexpression was induced with 1 mM IPTG, followed by incubation at 17˚C overnight.…”

Section: Expression and Purification Of Pepsmentioning

confidence: 99%

On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer

McCormick

Jakeman

2015

Biochem. Cell Biol.

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Phosphoenolpyruvate synthase (PEPs) catalyzes the conversion of pyruvate to phosphoenolpyruvate (PEP) using a two-step mechanism invoking a phosphorylated-His intermediate. Formation of PEP is an initial step in gluconeogenesis, and PEPs is essential for growth of Escherichia coli on 3-carbon sources such as pyruvate. The production of PEPs has also been linked to bacterial virulence and antibiotic resistance. As such, PEPs is of interest as a target for antibiotic development, and initial investigations of PEPs have indicated inhibition by sodium fluoride. Similar inhibition has been observed in a variety of phospho-transfer enzymes through the formation of metal fluoride complexes within the active site. Herein we quantify the inhibitory capacity of sodium fluoride through a coupled spectrophotometric assay. The observed inhibition provides indirect evidence for the formation of a MgF3(-) complex within the enzyme active site and insight into the phospho-transfer mechanism of PEPs. The effect of AlCl3 on PEPs enzyme activity was also assessed and found to decrease substrate binding and turnover.

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Section: Expression and Purification Of Pepsmentioning

confidence: 99%

On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer

McCormick

Jakeman

2015

Biochem. Cell Biol.

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“…Compounds 9 and 11 were first evaluated as Glc-1-P (1) analogue substrates for Cps2L given that the enzyme had previously demonstrated a broad substrate specificity, 34,37,38 including poor conversion (3−10%) observed for β-L-fucosyl phosphate. 34 Negligible turnover (<1%) to sugar nucleotide products were observed by HPLC over 24 h using 2−10 EU of Cps2L with a 8:1 ratio of 9 or 11 relative to dTTP substrate (2) (Supporting Information). This suggested that the L-rhamnose phosphonates did not act as substrates for Cps2L.…”

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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“…12 The recent observation that the glycosyltransferase VinC is capable of processing both a-and b-anomer of a sugar nucleotide to give glycosylated products with corresponding inversion of stereochemistry at the anomeric linkage 13 underlies the importance of stereospecific synthetic methods for the preparation of these substrates. Although the enzymatic preparation of these compounds has emerged, [14][15][16][17][18] the versatility available via chemical synthesis is unparalleled.…”

Section: Introductionmentioning

confidence: 99%

Stereospecific synthesis of sugar-1-phosphates and their conversion to sugar nucleotides

Timmons

Jakeman

2008

Carbohydrate Research

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Exploiting Nucleotidylyltransferases To Prepare Sugar Nucleotides

Cited by 49 publications

References 35 publications

On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer

On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer

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

Stereospecific synthesis of sugar-1-phosphates and their conversion to sugar nucleotides

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