1. Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus
- Author
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Kerstin Steiner, Gregor Hagelueken, Paul Messner, Christina Schäffer, James H. Naismith, The Wellcome Trust, University of St Andrews. School of Chemistry, University of St Andrews. Biomedical Sciences Research Complex, and University of St Andrews. EaSTCHEM
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Models, Molecular ,Family GT4 ,Plasma protein binding ,Glycogen-Synthase ,Mannans ,Geobacillus stearothermophilus ,chemistry.chemical_compound ,Structural Biology ,Catalytic Domain ,Deoxy Sugars ,Transferase ,QD ,SER, surface entropy reduction ,rhamnosyltransferase ,0303 health sciences ,biology ,ITC, isothermal titration calorimetry ,Nucleoside Diphosphate Sugars ,030302 biochemistry & molecular biology ,Biochemistry ,Rhamnosyltransferase ,Diffraction data ,Protein Binding ,SeMet, selenomethionine ,crystal structure ,Glycan ,Rhamnose ,S-layer protein glycosylation ,Catalysis ,Article ,03 medical and health sciences ,Sequence ,Glycosyltransferase ,Thymine Nucleotides ,Gene-cluster ,Binding site ,Molecular Biology ,030304 developmental biology ,Glycoprotein glycan Biosynthesis ,Binding Sites ,Crystal structure ,Glycosyltransferases ,Substrate (chemistry) ,QD Chemistry ,Protein Structure, Tertiary ,Amino Acid Substitution ,Hexosyltransferases ,chemistry ,Mutagenesis, Site-Directed ,biology.protein ,Mannosyltreansferase pima ,Mutant Proteins - Abstract
Carbohydrate polymers are medically and industrially important. The S-layer of many Gram-positive organisms comprises protein and carbohydrate polymers and forms an almost paracrystalline array on the cell surface. Not only is this array important for the bacteria but it has potential application in the manufacture of commercially important polysaccharides and glycoconjugates as well. The S-layer glycoprotein glycan from Geobacillus stearothermophilus NRS 2004/3a is mainly composed of repeating units of three rhamnose sugars linked by alpha-1,3-, alpha-1,2-, and beta-1,2-linkages. The formation of the beta-1,2-linkage is catalysed by the enzyme WsaF. The rational use of this system is hampered by the fact that WsaF and other enzymes in the pathway share very little homology to other enzymes. We report the structural and biochemical characterisation of WsaF, the first such rhamnosyltransferase to be characterised. Structural work was aided by the surface entropy reduction method. The enzyme has two domains, the N-terminal domain, which binds the acceptor (the growing rhamnan chain), and the C-terminal domain, which binds the substrate (dTDP-beta-L-rhamnose). The structure of WsaF bound to dTDP and dTDP-beta-L-rhamnose coupled to biochemical analysis identifies the residues that underlie catalysis and substrate recognition. We have constructed and tested by site-directed mutagenesis a model for acceptor recognition. (C) 2010 Elsevier Ltd. All rights reserved. Publisher PDF
- Published
- 2010
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