Svetlana Borisova

University of Texas at Austin, Austin, Texas, United States

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Publications (21)119.6 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The homologous human ABO(H) A and B blood group glycosyltransferases GTA and GTB have two mobile polypeptide loops surrounding their active sites that serve to allow substrate access and product egress and to recognize and sequester substrates for catalysis. Previous studies have established that these enzymes can move from the 'open' state to the 'semi-closed' then 'closed' states in response to addition of substrate. The contribution of electrostatic interactions to these conformational changes has now been demonstrated by the determination at various pH of the structures of GTA, GTB, and the chimeric enzyme ABBA. Near neutral pH, GTA displays the closed state in which both mobile loops order around the active site, whereas ABBA and GTB display the open state. At low pH the apparent protonation of the DXD motif in GTA leads to expulsion of the donor analog to yield the open state, while at high pH both ABBA and GTB form the semi-closed state in which the first mobile loop becomes an ordered α-helix. Step-wise deprotonation of GTB in increments of 0.5 between pH 6.5 and 10.0 shows that helix ordering is gradual, which indicates that the formation of the semi-closed state is dependent on electrostatic forces consistent with the binding of substrate. Spectropolarimetric studies of the corresponding stand-alone peptide in solution reveal no tendency towards helix formation from pH 7.0 to 10.0, which shows that pH-dependent stability is a product of the larger protein environment and underlines the importance of substrate in active site ordering.
    Glycobiology 11/2013; 24(3). DOI:10.1093/glycob/cwt098 · 3.15 Impact Factor
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    ABSTRACT: The human ABO(H) A and B blood group glycosyltransferases GTA and GTB differ by only four amino acids, yet this small dissimilarity is responsible for significant differences in biosynthesis, kinetics and structure. Like other glycosyltransferases, these two enzymes have been shown to recognize substrates through dramatic conformational changes in mobile polypeptide loops surrounding the active site. Structures of GTA, GTB and several chimeras determined by single-crystal X-ray diffraction demonstrate a range of susceptibility to the choice of cryoprotectant, in which the mobile polypeptide loops can be induced by glycerol to form the ordered closed conformation associated with substrate recognition and by MPD [hexylene glycol, (±)-2-methyl-2,4-pentanediol] to hinder binding of substrate in the active site owing to chelation of the Mn²⁺ cofactor and thereby adopt the disordered open state. Glycerol is often avoided as a cryoprotectant when determining the structures of carbohydrate-active enzymes as it may act as a competitive inhibitor for monosaccharide ligands. Here, it is shown that the use of glycerol as a cryoprotectant can additionally induce significant changes in secondary structure, a phenomenon that could apply to any class of protein.
    Acta Crystallographica Section D Biological Crystallography 03/2012; 68(Pt 3):268-76. DOI:10.1107/S0907444912001801 · 2.67 Impact Factor
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    ABSTRACT: Cell surface mucins configure the cell surface by presenting extended protein backbones that are heavily O-glycosylated. The glycopeptide structures establish physicochemical properties at the cell surface that enable and block the formation of biologically important molecular complexes. Some mucins, such as MUC1, associate with receptor tyrosine kinases and other cell surface receptors, and engage in signal transduction in order to communicate information regarding conditions at the cell surface to the nucleus. In that context, the MUC1 cytoplasmic tail (MUC1CT) receives phosphorylation signals from receptor tyrosine kinases and serine/threonine kinases, which enables its association with different signaling complexes that conduct these signals to the nucleus and perhaps other subcellular organelles. We have detected the MUC1CT at promoters of over 500 genes, in association with several different transcription factors, and have shown that promoter occupancy can vary under different growth factor conditions. However, the full biochemical nature of the nuclear forms of MUC1 and its function at these promoter regions remain undefined. I will present evidence that nuclear forms of the MUC1CT include extracellular and cytoplasmic tail domains. In addition, I will discuss evidence for a hypothesis that the MUC1CT possesses a novel catalytic function that enables remodeling of the transcription factor occupancy of promoters, and thereby engages in regulation of gene expression.
    Glycobiology 11/2011; 21(11):1454-531. DOI:10.1093/glycob/cwr126 · 3.15 Impact Factor
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    ABSTRACT: A divergent and highly stereoselective route to 11 glycosylated methymycin analogues has been developed. The key to the success of this method was the iterative use of the Pd-catalyzed glycosylation reaction and postglycosylation transformation. This unique application of Pd-catalyzed glycosylation demonstrates the breath of α/β- and d/l-glycosylation of macrolides that can be efficiently prepared using a de novo asymmetric approach to the carbohydrate portion.
    Organic Letters 10/2010; 12(22):5150-3. DOI:10.1021/ol102144g · 6.36 Impact Factor
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    Svetlana A Borisova · Hung-Wen Liu ·
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    ABSTRACT: The in vitro characterization of the catalytic activity of DesVII, the glycosyltransferase involved in the biosynthesis of the macrolide antibiotics methymycin, neomethymycin, narbomycin, and pikromycin in Streptomyces venezuelae, is described. DesVII is unique among glycosyltransferases in that it requires an additional protein component, DesVIII, for activity. Characterization of the metabolites produced by a S. venezuelae mutant lacking the desVIII gene confirmed that desVIII is important for the biosynthesis of glycosylated macrolides but can be replaced by at least one of the homologous genes from other pathways. The addition of recombinant DesVIII protein significantly improves the glycosylation efficiency of DesVII in the in vitro assay. When affinity-tagged DesVII and DesVIII proteins were coproduced in Escherichia coli, they formed a tight (αβ)(3) complex that is at least 10(3)-fold more active than DesVII alone. The formation of the DesVII/DesVIII complex requires coexpression of both genes in vivo and cannot be fully achieved by mixing the individual protein components in vitro. The ability of the DesVII/DesVIII system to catalyze the reverse reaction with the formation of TDP-desosamine was also demonstrated in a transglycosylation experiment. Taken together, our data suggest that DesVIII assists the folding of DesVII during protein production and remains tightly bound during catalysis. This requirement must be taken into consideration in the design of combinatorial biosynthetic experiments with new glycosylated macrolides.
    Biochemistry 09/2010; 49(37):8071-84. DOI:10.1021/bi1007657 · 3.02 Impact Factor
  • S. A. Borisova · S. R. Guppi · H. J. Kim · B. Wu · H.-w. Liu · G. A. O’Doherty ·
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    ABSTRACT: S. A. Borisova, S. R. Guppi, H. J. Kim, B. Wu, H.-w. Liu and G. A. O’Doherty, Org. Lett. 2010, 12, 5150–5153
    Organic Letters 01/2010; 12:5150. · 6.36 Impact Factor
  • Svetlana A Borisova · Hak Joong Kim · Xiaotao Pu · Hung-Wen Liu ·
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    ABSTRACT: Studies of the macrolide glycosyltransferase, DesVII/DesVIII, showed that when multiple hydroxyl groups are present, no regiospecificity of glycosylation is observed for the linear substrates. In contrast, when cyclic substrates with multiple glycosylation sites were tested, it displayed excellent regiospecificity in most cases. These results clearly indicate that this enzyme pair is a valuable tool for glycodiversification.
    ChemBioChem 07/2008; 9(10):1554-8. DOI:10.1002/cbic.200800155 · 3.09 Impact Factor
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    Chai-Lin Kao · Svetlana A Borisova · Hak Joong Kim · Hung-wen Liu ·
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    ABSTRACT: The two essential structural components of macrolide antibiotics are the polyketide aglycone and the appended sugars. The aglycone formation is catalyzed by polyketide synthase (PKS), and glycosylation is catalyzed by an appropriate glycosyltransferase. Although it has been shown that glycosylation occurs after the cyclic aglycone is released from PKS, it is not known whether the acyl carrier protein (ACP)-bound linear polyketide chain can also be processed by the corresponding glycosyltransferase. To explore this possibility, the aglycone, 10-deoxymethynolide, which is the precursor of methymycin and neomethymycin, was chemically synthesized in the linear form as a N-acetylcysteamine (NAC) thioester. Subsequent incubation with TDP-d-desosamine in the presence of the dedicated glycosyltransferase, DesVII, and activator, DesVIII, produces a more polar product whose high-resolution mass is consistent with the anticipated glycosylated product. This study demonstrated for the first time that a macrolide glycosyltransferase can also recognize and process the linear precursor of its macrolactone substrate with a reduced but measurable activity.
    Journal of the American Chemical Society 06/2006; 128(17):5606-7. DOI:10.1021/ja058433v · 12.11 Impact Factor
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    ABSTRACT: (Chemical Equation Presented) Two's Company: DesVII, a glycosyltransferase involved in the biosynthesis of macrolide antibiotics, is unusual in that it requires an additional protein partner, DesVIII, for its full activity. The level of substrate tolerance of the DesVII/DesVIII pair was explored.
    Angewandte Chemie International Edition 04/2006; 45(17):2748-53. DOI:10.1002/anie.200503195 · 11.26 Impact Factor
  • Svetlana A Borisova · Lishan Zhao · Charles E Melançon III · Chai-Lin Kao · Hung-Wen Liu ·
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    ABSTRACT: In vitro catalytic activity of DesVII, the glycosyltransferase involved in the biosynthesis of methymycin, neomethymycin, narbomycin, and pikromycin in Streptomyces venezuelae, is described. This is the first report of demonstrated in vitro activity of a glycosyltransferase involved in the biosynthesis of macrolide antibiotics. DesVII is unique among glycosyltransferases in that it requires an additional protein component, DesVIII, as well as basic pH for its full activity.
    Journal of the American Chemical Society 07/2004; 126(21):6534-5. DOI:10.1021/ja049967j · 12.11 Impact Factor
  • Lishan Zhao · Noelle J Beyer · Svetlana A Borisova · Hung-wen Liu ·
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    ABSTRACT: In our study of the biosynthesis of D-desosamine in Streptomyces venezuelae, we have cloned and sequenced the entire desosamine biosynthetic cluster. The deduced product of one of the genes, desR, in this cluster shows high sequence homology to beta-glucosidases, which catalyze the hydrolysis of the glycosidic linkages, a function not required for the biosynthesis of desosamine. Disruption of the desR gene led to the accumulation of glucosylated methymycin/neomethymycin products, all of which are biologically inactive. It is thus conceivable that methymycin/neomethymycin may be produced as inert diglycosides, and the DesR protein is responsible for transforming these antibiotics from their dormant to their active forms. This hypothesis is supported by the fact that the translated desR gene has a leader sequence characteristic of secretory proteins, allowing it to be transported through the cell membrane and hydrolyze the modified antibiotics extracellularly to activate them. Expression of desR and biochemical characterization of the purified protein confirmed the catalytic function of this enzyme as a beta-glycosidase capable of catalyzing the hydrolysis of glucosylated methymycin/neomethymycin produced by S. venezuelae. These results provide strong evidence substantiating glycosylation/deglycosylation as a likely self-resistance mechanism of S. venezuelae. However, further experiments have suggested that such a glycosylation/deglycosylation is only a secondary self-defense mechanism in S. venezuelae, whereas modification of 23S rRNA, which is the target site for methymycin and its derivatives, by PikR1 and PikR2 is a primary self-resistance mechanism. Considering that postsynthetic glycosylation is an effective means to control the biological activity of macrolide antibiotics, the availability of macrolide glycosidases, which can be used for the activation of newly formed antibiotics that have been deliberately deactivated by engineered glycosyltransferases, may be a valuable part of an overall strategy for the development of novel antibiotics using the combinatorial biosynthetic approach.
    Biochemistry 01/2004; 42(50):14794-804. DOI:10.1021/bi035501m · 3.02 Impact Factor
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    ABSTRACT: Blood group A and B antigens are carbohydrate structures that are synthesized by glycosyltransferase enzymes. The final step in B antigen synthesis is carried out by an alpha1-3 galactosyltransferase (GTB) that transfers galactose from UDP-Gal to type 1 or type 2, alphaFuc1-->2betaGal-R (H)-terminating acceptors. Similarly the A antigen is produced by an alpha1-3 N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine from UDP-GalNAc to H-acceptors. Human alpha1-3 N-acetylgalactosaminyltransferase and GTB are highly homologous enzymes differing in only four of 354 amino acids (R176G, G235S, L266M, and G268A). Single crystal x-ray diffraction studies have shown that the latter two of these amino acids are responsible for the difference in donor specificity, while the other residues have roles in acceptor binding and turnover. Recently a novel cis-AB allele was discovered that produced A and B cell surface structures. It had codons corresponding to GTB with a single point mutation that replaced the conserved amino acid proline 234 with serine. Active enzyme expressed from a synthetic gene corresponding to GTB with a P234S mutation shows a dramatic and complete reversal of donor specificity. Although this enzyme contains all four "critical" amino acids associated with the production of blood group B antigen, it preferentially utilizes the blood group A donor UDP-GalNAc and shows only marginal transfer of UDP-Gal. The crystal structure of the mutant reveals the basis for the shift in donor specificity.
    Journal of Biological Chemistry 04/2003; 278(14):12403-5. DOI:10.1074/jbc.M212002200 · 4.57 Impact Factor
  • Lishan Zhao · Hiroshi Yamase · Svetlana A. Borisova · Hung-wen Liu ·

    XXIst International Carbohydrate Symposium 2002; 04/2002
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    ABSTRACT: The three-dimensional structure of the lumenal domain of the lectin-like chaperone calnexin determined to 2.9 A resolution reveals an extended 140 A arm inserted into a beta sandwich structure characteristic of legume lectins. The arm is composed of tandem repeats of two proline-rich sequence motifs which interact with one another in a head-to-tail fashion. Identification of the ligand binding site establishes calnexin as a monovalent lectin, providing insight into the mechanism by which the calnexin family of chaperones interacts with monoglucosylated glycoproteins.
    Molecular Cell 10/2001; 8(3):633-44. DOI:10.1016/S1097-2765(01)00318-5 · 14.02 Impact Factor
  • Lishan Zhao · Svetlana Borisova · S M Yeung · Hung-wen Liu ·

    Journal of the American Chemical Society 09/2001; 123(32):7909-10. DOI:10.1021/ja010587x · 12.11 Impact Factor
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    ABSTRACT: Epitope mapping studies and the determination of the structure to 1.8 A resolution have been carried out for the antigen-binding fragment MR1 in complex with peptide antigen. MR1 is specific for the novel fusion junction of the mutant epidermal growth factor receptor EGFRvIII and has been reported to have a high degree of specificity for the mutant EGFRvIII over the wild-type EGF receptor. The structure of the complex shows that the peptide antigen residue side-chains found by epitope mapping studies to be critical for recognition are accommodated in pockets on the surface of the Fv. However, the most distinctive portion of the peptide antigen, the novel fusion glycine residue, makes no contact to the Fv and does not contribute directly to the epitope. The specificity of MR1 lies in the ability of this glycine residue to assume the restricted conformation needed to form a type II' beta-hairpin turn more easily, and demonstrates that a peptide antigen can be used to generate a conformational epitope.
    Journal of Molecular Biology 06/2001; 308(5):883-93. DOI:10.1006/jmbi.2001.4628 · 4.33 Impact Factor
  • Svetlana A. Borisova · Lishan Zhao · David H. Sherman · H W Liu ·
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    ABSTRACT: [formula: see text] The appended sugars in macrolide antibiotics are indispensable to the biological activities of these important drugs. In an effort to generate a set of novel macrolide derivatives, we have created a new analogue of methymycin and neomethymycin, antibiotics produced by Streptomyces venezuelae. This analogue 15 carrying a different sugar, D-quinovose, instead of D-desosamine, was constructed by taking advantage of targeted gene deletion combined with a specific pathway-independent C-3 reduction capability of the wild type S. venezuelae.
    Organic Letters 08/1999; 1(1):133-6. DOI:10.1021/ol9906007 · 6.36 Impact Factor
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    ABSTRACT: Calnexin is a molecular chaperone that facilitates folding of glycoproteins in the endoplasmic reticulum (ER). The cloned lumenal domain of canine calnexin, cnxDeltaTMC, retains its biological activity without the transmembrane and cytosolic region. For the purpose of structure determination we generated a crystallizable core by mild proteolysis and identified its termini by N-terminal sequencing and molecular mass determination. A truncated gene was cloned accordingly. Its product, cnxDeltaN25C15, was purified to apparent homogeneity and crystallized. This truncated variant remains biologically active as shown by its binding to monoglucosylated oligosaccharides and functional interaction with ERp57. A heavy atom derivative was identified.
    Journal of Structural Biology 12/1998; 123(3):260-4. DOI:10.1006/jsbi.1998.4032 · 3.23 Impact Factor
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    ABSTRACT: The activated 65 kDa lepidopteran-specific CryIA(a) toxin from the commercially most important strainBacillus thuringiensisvar.kurstakiHD-1 has been investigated by X-ray diffraction and for its ability to form channels in planar lipid bilayers. Its three-dimensional structure has been determined by a multiple isomorphous replacement method and refined at 2.25 Å resolution to anR-factor of 0.168 for data withI2δ(I). The toxin is made of three distinct domains. The N-terminal domain is a bundle of eight α-helices with the central, relatively hydrophobic helix surrounded by amphipathic helices. The middle and C-terminal domains contain mostly β-sheets. Comparison with the structure of CryIIIA, a coleopteran-specific toxin, shows that although the fold of these two proteins is similar, there are significant structural differences within domain II. This finding supports the conclusions from genetic studies that domain II is involved in recognition and binding to cell surface receptors. The distribution of electrostatic potential on the surface of the molecule is non-uniform and identifies one side of the α-helical domain as negatively charged. The predominance of arginine residues as basic residues ensures that the observed positive charge distribution is also maintained in the highly alkaline environment found in the lepidopteran midgut. Structurally important salt bridges that are conserved across Cry sequences were identified and their possible role in toxin action was postulated. In planar lipid bilayers, CryIA(a) forms cation-selective channels, whose conductance is significantly smaller than that reported for CryIIIA but similar to those of other Cry toxins.
    Journal of Molecular Biology 12/1995; 254(3-254):447-464. DOI:10.1006/jmbi.1995.0630 · 4.33 Impact Factor
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    ABSTRACT: A trypsin-activated CrylA(a) protein from Bacillus thuringiensis has been purified and crystallized. Crystals belong to orthorhombic space group P212121, with cell dimensions a = 53.3, b = 111.3 and c = 154.7 Å. The crystals diffract to at least 2.2 Å resolution and are suitable for X-ray structural analysis. They contain a single molecule in the asymmetric unit.
    Journal of Molecular Biology 11/1994; 243(3-243):530-532. DOI:10.1006/jmbi.1994.1678 · 4.33 Impact Factor

Publication Stats

1k Citations
119.60 Total Impact Points


  • 2001-2010
    • University of Texas at Austin
      • • Department of Chemistry and Biochemistry
      • • Division of Medicinal Chemistry
      Austin, Texas, United States
  • 2004
    • Concordia University Texas
      Austin, Texas, United States
  • 2001-2003
    • University of Ottawa
      • Department of Biochemistry, Microbiology and Immunology
      Ottawa, Ontario, Canada
  • 1999
    • University of Minnesota Duluth
      • Department of Chemistry and Biochemistry
      Duluth, Minnesota, United States
  • 1998
    • Montreal Heart Institute
      Montréal, Quebec, Canada
  • 1995
    • National Research Council Canada
      • Biotechnology Research Institute (BRI)
      Ottawa, Ontario, Canada