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ABSTRACT: Herein we report the first synthesis of a 3-deoxy-D-manno-octulosonic acid (KDO) building block starting from glucose through pathway engineering of Escherichia coli and subsequent chemical modifications to provide an alternative method to produce KDO, found in plant and bacterial oligosaccharides.
Organic & Biomolecular Chemistry 04/2012; 10(30):5856-60. · 3.70 Impact Factor
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ABSTRACT: A detailed understanding of the molecular mechanism of chaperone-assisted protein quality control is often hampered by the lack of well-defined homogeneous glycoprotein probes. We describe here a highly convergent chemoenzymatic synthesis of the monoglucosylated glycoforms of bovine ribonuclease (RNase) as specific ligands of lectin-like chaperones calnexin (CNX) and calreticulin (CRT) that are known to recognize the monoglucosylated high-mannose oligosaccharide component of glycoproteins in protein folding. The synthesis of a selectively modified glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase was accomplished by chemical synthesis of a large N-glycan oxazoline and its subsequent enzymatic ligation to GlcNAc-RNase under the catalysis of a glycosynthase. Selective removal of the terminal galactose by a β-galactosidase gave the Glc(1)Man(9)GlcNAc(2)-RNase glycoform in excellent yield. CD spectroscopic analysis and RNA-hydrolyzing assay indicated that the synthetic RNase glycoforms maintained essentially the same global conformations and were fully active as the natural bovine ribonuclease B. SPR binding studies revealed that the Glc(1)Man(9)GlcNAc(2)-RNase had high affinity to lectin CRT, while the synthetic Man(9)GlcNAc(2)-RNase glycoform and natural RNase B did not show CRT-binding activity. These results confirmed the essential role of the glucose moiety in the chaperone molecular recognition. Interestingly, the galactose-masked glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase also showed significant affinity to lectin CRT, suggesting that a galactose β-1,4-linked to the key glucose moiety does not significantly block the lectin binding. These synthetic homogeneous glycoprotein probes should be valuable for a detailed mechanistic study on how molecular chaperones work in concert to distinguish between misfolded and folded glycoproteins in the protein quality control cycle.
Journal of the American Chemical Society 08/2011; 133(36):14404-17. · 9.91 Impact Factor
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ABSTRACT: Microbacterium nematophilum causes a deleterious infection of the C. elegans hindgut initiated by adhesion to rectal and anal cuticle. C. elegans bus-2 mutants, which are resistant to M. nematophilum and also to the formation of surface biofilms by Yersinia sp., carry genetic lesions in a putative glycosyltransferase containing conserved domains of core-1 beta1,3-galactosyltransferases. bus-2 is predicted to act in the synthesis of core-1 type O-glycans. This observation implies that the infection requires the presence of host core-1 O-glycoconjugates and is therefore carbohydrate-dependent. Chemical analysis reported here reveals that bus-2 is indeed deficient in core-1 O-glycans. These mutants also exhibit a new subclass of O-glycans whose structures were determined by high performance tandem mass spectrometry; these are highly fucosylated and have a novel core that contains internally linked GlcA. Lectin studies showed that core-1 glycans and this novel class of O-glycans are both expressed in the tissue that is infected in the wild type worms. In worms having the bus-2 genetic background, core-1 glycans are decreased, whereas the novel fucosyl O-glycans are increased in abundance in this region. Expression analysis using a red fluorescent protein marker showed that bus-2 is expressed in the posterior gut, cuticle seam cells, and spermatheca, the first two of which are likely to be involved in secreting the carbohydrate-rich surface coat of the cuticle. Therefore, in the bus-2 background of reduced core-1 O-glycans, the novel fucosyl glycans likely replace or mask remaining core-1 ligands, leading to the resistance phenotype. There are more than 35 Microbacterium species, some of which are pathogenic in man. This study is the first to analyze the biochemistry of adhesion to a host tissue by a Microbacterium species.
Journal of Biological Chemistry 04/2010; 285(23):17662-72. · 4.77 Impact Factor
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ABSTRACT: Genome sequencing projects are suggesting there are dozens of glycosidase sequences that could be used to fingerprint cell types and serve as starting points for biocatalyst discovery. Herein, we present a simple chemical proteomics approach to profile intracellular glycosidase activities of three different bacterial cell extracts using a synthetic α- and β-linked library of 18 representative substrates with electrospray ionization-mass spectrometry (ESI-MS) reaction monitoring. Three target bacteria – Escherichia coli K12, Bacillus cereus and Pseudomonas aeruginosa – can be easily differentiated by this method. Compared with traditional chromogenic and fluorogenic methods to profile bacterial enzyme activities individually, this MS-based method can detect multiple enzyme activities in one reaction and easily highlight activity differences between whole cell extracts.
07/2009; 26(1-2):25-31.
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ABSTRACT: Herein we present an analysis of the chemical function of a recombinant bifunctional phosphomannose isomerase/GDP-mannose pyrophosphorylase (manC) from Pyrococcus furiosus DSM 3638 and its use in the synthesis of guanidinediphospho-hexoses and a range of nucleotidediphospho-mannoses. This enzyme is unusually promiscuous in both its nucleotide triphosphate (NTP) and sugar-1-phosphate acceptance. It accepts all five naturally occurring NTPs (ATP, CTP, GTP, dTTP and UTP) and a range of sugar-1-phosphates (glucose-, mannose-, galactose-, glucosamine-, N-acetylglucosamine- and fucose-1-phosphate). A truncated GDP-mannose pyrophosphorylase domain of the whole length enzyme showed almost 100-fold less sugar nucleotidyltransferase activity with only GTP and mannose 1-phosphate as substrates. The temperature stability and inherently broad substrate tolerance of this archaeal enzyme make it an effective reagent for the rapid chemoenzymatic synthesis of a range of natural and unnatural sugar nucleotides that are challenging to make by chemical means alone.
Organic & Biomolecular Chemistry 06/2009; 7(10):2135-9. · 3.70 Impact Factor
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ABSTRACT: Alpha-glucan phosphorylase catalyzes the reversible cleavage of alpha-1-4-linked glucose polymers into alpha-D-glucose-1-phosphate. We report the recombinant production of an alpha-glucan/maltodextrin phosphorylase (PF1535) from a hyperthermophilic archaeon, Pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. The apparent 98 kDa recombinant enzyme was active over a broad range of temperatures and pH, with optimal activity at 80 degrees C and pH 6.5-7. This archaeal protein retained its complete activity after 24 h at 80 degrees C in Tris-HCl buffer. Unlike other previously reported phosphorylases, the Ni-affinity column purified enzyme showed broad substrate specificity in both the synthesis and degradation of maltooligosaccharides. In the synthetic direction of the enzymatic reaction, the lowest oligosaccharide required for the chain elongation was maltose. In the degradative direction, the archaeal enzyme can produce glucose-1-phosphate from maltotriose or longer maltooligosaccharides including both glycogen and starch. The specific activity of the enzyme at 80 degrees C in the presence of 10 mM maltoheptaose and at 10 mg ml(-1) glycogen concentration was 52 U mg(-1) and 31 U mg(-1), respectively. The apparent Michaelis constant and maximum velocity for inorganic phosphate were 31 +/- 2 mM and 0.60 +/- 0.02 mM min(-1) microg(-1), respectively. An initial velocity study of the enzymatic reaction indicated a sequential bi-bi catalytic mechanism. Unlike the more widely studied mammalian glycogen phosphorylase, the Pyrococcus enzyme is active in the absence of added AMP.
Archaea (Vancouver, B.C.) 01/2009; 2(3):169-76.
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ABSTRACT: Neisseria meningitidis is a cause of fatal sepsis and epidemic meningitis. A major virulence factor is cell wall lipooligosaccharide (LOS). The M986 strain has been used extensively in immunological and vaccine research. Yet, the LOS repertoire of this strain is not known. Here we have investigated the LOS structures of M986 and three of its variants OP1, OP2-, and OP2+. This strain and its variants present a series of related LOS families that are increasingly truncated in their listed order. The major structural differences are seen in the lacto-N-neotetraose alpha-chain. The gamma-chain Hep II contains two phosphoethanolamine (PEA) substitutions at C3 and C6/7. These substitutions were seen in all strains except OP2+ where the canonical core Hep II is missing. The PEA disubstitution was present in nearly stoichiometric amounts with only minor amounts of monosubstitution observed, and no glycomers devoid of PEA were seen. This was also the case in LOS with a complete lacto-N-neotetraosyl alpha-chain even though previous reports suggested that the presence of an extended alpha-chain hinders C3 PEA substitution of Hep II. Approximately 50% of gamma-chain GlcNAc was present in its 3-OAc-substituted form. Because Hep II C3 PEA substitution and gamma-chain GlcNAc OAc addition have been reported to negatively interact, the co-existence of these two modifications in these strains is unique. The LOS structures of M986 and three of its variants have been determined, which better defines these strains as tools for immunological and vaccine research.
Journal of Biological Chemistry 01/2009; 284(7):4616-25. · 4.77 Impact Factor
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ABSTRACT: In this study, we report the cloning, recombinant expression, and biochemical characterization of a heat-stable CMP-N-acylneuraminic acid (NeuAc) synthetase from Clostridium thermocellum ATCC 27405. A high throughput electrospray ionization mass spectrometry (ESI-MS)-based assay demonstrates that the enzyme has an absolute requirement for a divalent cation for activity and reaches maximum activity in the presence of 10 mM Mn(2+). The enzyme is active at pH 8-13 in Tris-HCl buffer and at 37-60 degrees C, and maximum activity is observed at pH 9.5 and 50 degrees C in the presence of 0.2 mM dithiothreitol. In addition to NeuAc, the enzyme also accepts the analog N-glycolylneuraminic acid (NeuGc) as a substrate. The apparent Michaelis constants for cytidine triphosphate and NeuAc or NeuGc are 240 +/- 20, 130 +/- 10, and 160 +/- 10 microM, respectively, with corresponding turnover numbers of 3.33, 2.25, and 1.66 s(-1), respectively. An initial velocity study of the enzymatic reaction indicates an ordered bi-bi catalytic mechanism. In addition to demonstration of a thermostable and substrate-tolerant enzyme, confirmation of the biochemical function of a gene for CMP-NeuAc synthetase in C. thermocellum also opens the question of the biological function of CMP-NeuAc in such nonpathogenic microorganisms.
Applied Microbiology and Biotechnology 10/2007; 76(4):827-34. · 3.42 Impact Factor
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ABSTRACT: Herein we present the chemical function analysis of a recombinant sugar nucleotidyltransferase from the hyperthermophile Pyrococcus furiosus and its use in the one-pot synthesis of chloroacetyl- and alkyne-tagged analogues of uridinediphospho-N-acetylglucosamine (UDP-GlcNAc). The gene was originally annotated as a glucose-1-phosphate deoxythymidylyltransferase; however, kinetic analysis of a panel of sugar-1-phosphates with the protein shows that it is better described as a bifunctional protein that synthesizes UDP-GlcNAc from glucosamine-1-phosphate and acetyl coenzyme A (CoA). A new mass-spectrometry-based assay for the rapid analysis of the acyltransferase activity demonstrates that the enzyme can also accept cheaper truncated N-acetylcysteamine thioester substrates in place of the natural acetyl CoA. The enzyme can tolerate alkyne or chloride substitutions in the acyl moiety, thereby allowing the facile synthesis of tagged sugar nucleotides for future use in protein O-GlcNAc modification studies.
Journal of the American Chemical Society 02/2005; 127(3):836-7. · 9.91 Impact Factor
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ABSTRACT: Herein, we report the first cloning, recombinant expression, and synthetic utility of a sugar nucleotidyltransferase from any archaeal source and demonstrate by an electrospray ionization mass spectrometry (ESI-MS)-based assay its unusual tolerance of heat, pH, and sugar substrates. The metal-ion-dependent enzyme from Pyrococcus furiosus DSM 3638 showed a relatively high degree of acceptance of glucose-1-phosphate (Glc1P), mannose-1-phosphate (Man1P), galactose-1-phosphate (Gal1P), fucose-1-phosphate, glucosamine-1-phosphate, galactosamine-1-phosphate, and N-acetylglucosamine-1-phosphate with uridine and deoxythymidine triphosphate (UTP and dTTP, respectively). The apparent Michaelis constants for Glc1P, Man1P, and Gal1P are 13.0 +/- 0.7, 15 +/- 1, and 22 +/- 2 microM, respectively, with corresponding turnover numbers of 2.08, 1.65, and 1.32 s(-1), respectively. An initial velocity study indicated an ordered bi-bi catalytic mechanism for this enzyme. The temperature stability and inherently broad substrate tolerance of this archaeal enzyme promise an effective reagent for the rapid chemoenzymatic synthesis of a range of natural and unnatural sugar nucleotides for in vitro glycosylation studies and highlight the potential of archaea as a source of new enzymes for synthesis.
Journal of the American Chemical Society 01/2005; 126(49):15993-8. · 9.91 Impact Factor
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ABSTRACT: UDP-α-d-glucose pyrophosphorylase (α-d-glucose-1-phosphate uridyltransferase) (UGPase) catalyzes the formation of activated UDP-glucose from glucose-1-phosphate and UTP. Herein we report the cloning and characterization of a thermostable UGPase from a mesophilic, Gram-negative pathogen Helicobacter pylori. An electrospray ionization mass spectrometry (ESI-MS)-based assay demonstrates that the recombinant enzyme absolutely requires divalent cation for its activity, reaching a maximum in the presence of 3 mM Mg2+. The optimum activity of the enzyme was around pH 8–9.5 in Tris–HCl buffer and at 37–70 °C; maximum activity was observed at pH 8.5 and 60 °C. Apart from its natural substrate, glucose-1-phosphate, the enzyme also accepts mannose-, galactose-, and glucosamine-1-phosphates. The apparent Michaelis constants of the purified enzyme for glucose-1-phosphate with UTP and dTTP and for mannose-1-phosphate with UTP are 15 ± 2, 32 ± 4 and 77 ± 9 μM, respectively, with the corresponding turnover numbers 5.4, 5.4 and 1.2 min−1, respectively. An initial velocity study of the forward reaction of the enzyme indicates an ordered bi–bi catalytic mechanism. Analysis of the genomes of other organisms that grow at 37 °C predicts many more such thermostable biocatalysts.
Journal of Molecular Catalysis B: Enzymatic.
