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Gilbert, Michel; Watson, David; Wakarchuk, Warren W.

doi:10.1023/a:1018379607492

Cited by 20 publications

(10 citation statements)

References 11 publications

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“…Using the continuous assay method, we determined the steady‐state kinetic parameters for CTP at a fixed Neu5Ac concentration of 3.0 m M (see Figure S3, Supporting Information) with a K M value of 54±8 μM, which is almost identical to a very recently published value of 34±5 μM using a different continuous assay method 11. In contrast, these values are about one order of magnitude lower than data determined earlier by using different discontinuous assay methods (0.56 mM, [12] 0.31 mM,18 0.17 mM [8] ). For CTP k cat was determined at 58.9±2.1 s −1 and the catalytic efficiency k cat /K M was 1090.7 mM −1 s −1 .…”

Section: Resultssupporting

confidence: 82%

“…Most methods for assaying CSS activity reported so far are discontinuous and require quenching of the catalytic reaction at discrete time intervals, such as for radiolabel based quantification,12,13 the common TBA assay14 or its variations,15 as well as for coupled enzyme assays that measure converted or remaining sialic acid via NADH consumption,16 or by assays for the released inorganic pyrophosphate 8. Alternatively, CMP‐activated product formation can be quantified by HPLC,17 capillary electrophoresis18,19 or ESI‐MS assays20 although the latter methods are rarely used due to the high cost of instrumentation. The general problem of discontinuous assays is not only that these are rather time consuming and laborious, but that data points are often gathered outside of initial rate conditions.…”

Section: Introductionmentioning

confidence: 99%

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Flexibility of Substrate Binding of Cytosine‐5′‐Monophosphate‐N‐Acetylneuraminate Synthetase (CMP‐Sialate Synthetase) from Neisseria meningitidis: An Enabling Catalyst for the Synthesis of Neo‐sialoconjugates

Dong

Fessner

2011

Adv Synth Catal

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Abstract:We have successfully established a simple continuous colorimetric assay for the sensitive and reliable quantification of cytosine-5'-monophosphate-acetylneuraminate synthetase (CMP-sialate synthetase, CSS) activity based on the pH change of a released proton equivalent upon nucleotide activation of Neu5Ac or related analogues. Using this method, steady-state kinetic data of Neisseria meningitidis CSS were determined for cytosine-5'-triphosphate (CTP), N-acetylneuraminic acid (Neu5Ac) and eleven structural variations of the sialic acid, including backbone truncation, deamination, epimerization, and several N-acyl modifications. These data further demonstrate the unusual versatility of the N. meningitidis CSS for a general access to sialoconjugates containing non-natural sialic acid analogues. Remarkably, the assay allows covering a broad range of substrate parameters that span over more than three orders of magnitude for K M and k cat measurements. With the aid of a structural model built from X-ray crystal structure data, the kinetic data could be used to interpret potential protein contributions in substrate binding of Neu5Ac and its analogues. The Neu5Ac analogues were used for the preparation of neo-sialoconjugate analogues of 2,6-sialyllactose in a one-pot two-enzyme cascade, using N. meningitidis CSS together with the novel a2,6-sialyltransferase (a2,6SiaT) from Photobacterium leiognathi, which proved to be highly effective because of its optimum activity at alkaline pH, appropriately matching the requirements from the overall reaction system.

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Section: Resultssupporting

confidence: 82%

Section: Introductionmentioning

confidence: 99%

Flexibility of Substrate Binding of Cytosine‐5′‐Monophosphate‐N‐Acetylneuraminate Synthetase (CMP‐Sialate Synthetase) from Neisseria meningitidis: An Enabling Catalyst for the Synthesis of Neo‐sialoconjugates

Dong

Fessner

2011

Adv Synth Catal

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“…[24] Deletion mutants nanA À were screened for their inability to grow on M9 medium with N-acetylneuraminic acid as the sole carbon and energy source. To obtain the plasmid pBS-nsyt, the XbaI-XbaI fragment containing the CMP-NeuAc synthase gene was excised from the plasmid pNSY-01 [25] and cloned into the XbaI site of plasmid pNST01 [26] upstream from the a-2,3-sialyltransferase gene. The plasmid pBBRwbpP was constructed by inserting the XbaI-EcoRI fragment from pFV617 ± 26a, [17] which contains the UDP-GlcNAc-4-epimerase gene, into the XbaIEcoRI sites of pBBR1MCS-2.…”

Section: Methodsmentioning

confidence: 99%

Large‐Scale In Vivo Synthesis of the Carbohydrate Moieties of Gangliosides GM1 and GM2 by Metabolically Engineered Escherichia coli

et al. 2003

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Two metabolically engineered Escherichia coli strains have been constructed to produce the carbohydrate moieties of gangliosides GM2 (GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc; Gal = galactose, Glc = glucose, Ac = acetyl) and GM1 (Galbeta-3GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc. The GM2 oligosaccharide-producing strain TA02 was devoid of both beta-galactosidase and sialic acid aldolase activities and overexpressed the genes for CMP-NeuAc synthase (CMP = cytidine monophosphate), alpha-2,3-sialyltransferase, UDP-GlcNAc (UDP = uridine diphosphate) C4 epimerase, and beta-1,4-GalNAc transferase. When this strain was cultivated on glycerol, exogenously added lactose and sialic acid were shown to be actively internalized into the cytoplasm and converted into GM2 oligosaccharide. The in vivo synthesis of GM1 oligosaccharide was achieved by taking a similar approach but using strain TA05, which additionally overexpressed the gene for beta-1,3-galactosyltransferase. In high-cell-density cultures, the production yields for the GM2 and GM1 oligosaccharides were 1.25 g L(-1) and 0.89 g L(-1), respectively.

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“…Protein Purification-Recombinant CMP-NeuAc synthetase was overexpressed in E. coli and purified using published protocols (18) with modifications. Briefly we used a combination of anion-exchange on a MonoQ column (Amersham Pharmacia Biotech) and gel filtration on a Superose 12 column (Amersham Pharmacia Biotech).…”

Section: Methodsmentioning

confidence: 99%

Structure of a Sialic Acid-activating Synthetase, CMP-acylneuraminate Synthetase in the Presence and Absence of CDP

Mosimann

Gilbert²,

Dombroswki

et al. 2001

Journal of Biological Chemistry

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The x-ray crystallographic structure of selenomethionyl cytosine-5-monophosphate-acylneuraminate synthetase (CMP-NeuAc synthetase) from Neisseria meningitidis has been determined at 2.0-Å resolution using multiple-wavelength anomalous dispersion phasing, and a second structure, in the presence of the substrate analogue CDP, has been determined at 2.2-Å resolution by molecular replacement. This work identifies the active site residues for this class of enzyme for the first time. The detailed interactions between the enzyme and CDP within the mononucleotide-binding pocket are directly observed, and the acylneuraminate-binding pocket has also been identified. A model of acylneuraminate bound to CMP-NeuAc synthetase has been constructed and provides a structural basis for understanding the mechanism of production of "activated" sialic acids. Sialic acids are key saccharide components on the surface of mammalian cells and can be virulence factors in a variety of bacterial species (e.g. Neisseria, Haemophilus, group B streptococci, etc.). As such, the identification of the bacterial CMP-NeuAc synthetase active site can serve as a starting point for rational drug design strategies.Cytosine-5Ј-monophosphate-acylneuraminate synthetase (cytosine-5Ј-monophosphate-N-acetyl neuraminic acid synthetase, CMP-NeuAc synthetase) 1 catalyzes the penultimate step in the addition of sialic acids to the oligosaccharide component of glycoconjugates and is a required component of sialylation pathways. CMP-NeuAc synthetase-deficient mutants do not express sialylated glycoconjugates and can be complemented with functional CMP-NeuAc synthetase in both mammalian (1) and bacterial systems (2). Sialic acids participate in a myriad of signaling, recognition, and cell-cell adhesion phenomena in mammalian cells (3) and are overexpressed in some highly malignant tumors (4). Genetic disorders that lead to altered physiological levels of sialic acids have many consequences in humans and can be fatal (5). In bacterial systems, sialic acids are less common and frequently have roles as virulence factors (6). In Neisseria gonorrhoeae, sialylated glycoconjugates protect the organism from phagocytosis and increase serum resistance (7). In Haemophilus ducreyi the presence of 2-keto-3-deoxy-manno-octulosonate-containing glycoconjugates correlate with the organism's pathogenicity (8). Likewise, the sialylated capsule of Neisseria meningitidis, Escherichia coli K1, and group B streptococci are virulence factors (9-11). It has been suggested that bacterial species produce sialylated glycoconjugates to mimic the host and escape detection by the immune system (12). Clearly, the mechanism of production of sialic acid-containing glycoconjugates is of clinical interest. Although bacterial and eucaryotic CMPNeuAc synthetase enzymes share many catalytic properties, several important differences have been reported, including substrate specificity, tertiary structure, inhibitor sensitivity, and cellular localization (13). As a result, bacterial CMP-NeuAc synthetase en...

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Untitled

Cited by 20 publications

References 11 publications

Flexibility of Substrate Binding of Cytosine‐5′‐Monophosphate‐N‐Acetylneuraminate Synthetase (CMP‐Sialate Synthetase) from Neisseria meningitidis: An Enabling Catalyst for the Synthesis of Neo‐sialoconjugates

Flexibility of Substrate Binding of Cytosine‐5′‐Monophosphate‐N‐Acetylneuraminate Synthetase (CMP‐Sialate Synthetase) from Neisseria meningitidis: An Enabling Catalyst for the Synthesis of Neo‐sialoconjugates

Large‐Scale In Vivo Synthesis of the Carbohydrate Moieties of Gangliosides GM1 and GM2 by Metabolically Engineered Escherichia coli

Structure of a Sialic Acid-activating Synthetase, CMP-acylneuraminate Synthetase in the Presence and Absence of CDP

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