Enzymatic Synthesis of Fluorinated Mechanistic Probes for Sialidases and Sialyltransferases

Department of Chemistry, University of California, One Shields Avenue, Davis, California 95616, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 10/2007; 129(35):10630-1. DOI: 10.1021/ja072687u
Source: PubMed


A general, convenient, and efficient enzymatic approach for producing fluorinated mechanistic probes for sialidases and sialyltransferases is described. Other than the reported sialyltransferase inhibitor CMP-3F(axial)Neu5Ac, CIVIP-3F (equatorial)Sia derivatives (in which Sia is Neu5Ac or Neu5Gc) have been synthesized along with CMP-3F (axial)Neu5Gc and CMP-3F(axia/)KDN as novel inhibitors and important mechanistic probes for sialyltransferases. 3-Fluoro-sialosides have been enzymatically synthesized and could be used as potential inhibitors and mechanistic probes for sialidases and as probes for sialic acid-binding proteins.

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    • "' stands for the axial position of the fluorine at carbon 3) was obtained by soaking with 1.25 mM of CMP-3F(a)Neu5Ac (final concentration) for 30 min prior to cryocooling and data collection. The CMP-3F(a)Neu5Ac analog was synthesized as previously described [16] [28]. The crystals were placed in a reservoir solution containing 20% ethylene glycol (for both crystals of D15Pd2,6ST(N)) or Paratone-N oil (for crystal of D112Pd2,6ST(N)) and flash cooled in liquid nitrogen prior to data collection. "
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    ABSTRACT: Sialyltransferase structures fall into either GT-A or GT-B glycosyltransferase fold. Some sialyltransferases from the Photobacterium genus have been shown to contain an additional N-terminal immunoglobulin (Ig)-like domain. Photobacterium damselae α2-6-sialyltransferase has been used efficiently in enzymatic and chemoenzymatic synthesis of α2-6-linked sialosides. Here we report three crystal structures of this enzyme. Two structures with and without a donor substrate analog CMP-3F(a)Neu5Ac contain an immunoglobulin (Ig)-like domain and adopt the GT-B sialyltransferase fold. The binary structure reveals a non-productive pre-Michaelis complex, which are caused by crystal lattice contacts that prevent the large conformational changes. The third structure lacks the Ig-domain. Comparison of the three structures reveals small inherent flexibility between the two Rossmann-like domains of the GT-B fold. Copyright © 2014. Published by Elsevier B.V.
    FEBS Letters 11/2014; 588(24). DOI:10.1016/j.febslet.2014.11.003 · 3.17 Impact Factor
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    • "CMP-sialic acids and α2–3-and α2–6-linked sialosides containing 3F(axial)-sialic acid or 3F(equatorial)-sialic acid residue have also been obtained using a one-pot two-enzyme system containing a sialyltransferase and NmCSS from purified 3F(axial)-sialic acid or 3F(equatorial)-sialic acid synthesized by an E. coli sialic acid-catalyzed reaction (Chokhawala et al. 2007a). "
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    ABSTRACT: Sialic acids are a family of negatively charged monosaccharides which are commonly presented as the terminal residues in glycans of the glycoconjugates on eukaryotic cell surface or as components of capsular polysaccharides or lipooligosaccharides of some pathogenic bacteria. Due to their important biological and pathological functions, the biosynthesis, activation, transfer, breaking down, and recycle of sialic acids are attracting increasing attention. The understanding of the sialic acid metabolism in eukaryotes and bacteria leads to the development of metabolic engineering approaches for elucidating the important functions of sialic acid in mammalian systems and for large-scale production of sialosides using engineered bacterial cells. As the key enzymes in biosynthesis of sialylated structures, sialyltransferases have been continuously identified from various sources and characterized. Protein crystal structures of seven sialyltransferases have been reported. Wild-type sialyltransferases and their mutants have been applied with or without other sialoside biosynthetic enzymes for producing complex sialic acid-containing oligosaccharides and glycoconjugates. This mini-review focuses on current understanding and applications of sialic acid metabolism and sialyltransferases.
    Applied Microbiology and Biotechnology 04/2012; 94(4):887-905. DOI:10.1007/s00253-012-4040-1 · 3.34 Impact Factor
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    ABSTRACT: Sialic acids are abundant nine-carbon sugars expressed terminally on glycoconjugates of eukaryotic cells and are crucial for a variety of cell biological functions such as cell-cell adhesion, intracellular signaling, and in regulation of glycoproteins stability. In bacteria, N-acetylneuraminic acid (Neu5Ac) polymers are important virulence factors. Cytidine 5'-monophosphate (CMP)-N-acetylneuraminic acid synthetase (CSS; EC, the key enzyme that synthesizes CMP-N-acetylneuraminic acid, the donor molecule for numerous sialyltransferase reactions, is present in both prokaryotes and eukaryotic systems. Herein, we emphasize the source, function, and biotechnological applications of CSS enzymes from bacterial sources. To date, only a few CSS from pathogenic bacterial species such as Neisseria meningitidis, Escherichia coli, group B streptococci, Haemophilus ducreyi, and Pasteurella hemolytica and an enzyme from nonpathogenic bacterium, Clostridium thermocellum, have been described. Overall, the enzymes from both Gram-positive and Gram-negative bacteria share common catalytic properties such as their dependency on divalent cation, temperature and pH profiles, and catalytic mechanisms. The enzymes, however, can be categorized as smaller and larger enzymes depending on their molecular weight. The larger enzymes in some cases are bifunctional; they have exhibited acetylhydrolase activity in addition to their sugar nucleotidyltransferase activity. The CSSs are important enzymes for the chemoenzymatic synthesis of various sialooligosaccharides of significance in biotechnology.
    Applied Microbiology and Biotechnology 09/2008; 80(5):757-65. DOI:10.1007/s00253-008-1643-7 · 3.34 Impact Factor
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