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Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus

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...

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Autores principales: Steiner, Kerstin, Hagelueken, Gregor, Messner, Paul, Schäffer, Christina, Naismith, James H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898925/
https://www.ncbi.nlm.nih.gov/pubmed/20097205
http://dx.doi.org/10.1016/j.jmb.2010.01.035
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author Steiner, Kerstin
Hagelueken, Gregor
Messner, Paul
Schäffer, Christina
Naismith, James H.
author_facet Steiner, Kerstin
Hagelueken, Gregor
Messner, Paul
Schäffer, Christina
Naismith, James H.
author_sort Steiner, Kerstin
collection PubMed
description 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 α-1,3-, α-1,2-, and β-1,2-linkages. The formation of the β-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-β-l-rhamnose). The structure of WsaF bound to dTDP and dTDP-β-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.
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spelling pubmed-38989252014-01-24 Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus Steiner, Kerstin Hagelueken, Gregor Messner, Paul Schäffer, Christina Naismith, James H. J Mol Biol Article 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 α-1,3-, α-1,2-, and β-1,2-linkages. The formation of the β-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-β-l-rhamnose). The structure of WsaF bound to dTDP and dTDP-β-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. Elsevier 2010-03-26 /pmc/articles/PMC3898925/ /pubmed/20097205 http://dx.doi.org/10.1016/j.jmb.2010.01.035 Text en © 2010 Elsevier Ltd. https://creativecommons.org/licenses/by/3.0/ Open Access under CC BY 3.0 (https://creativecommons.org/licenses/by/3.0/) license
spellingShingle Article
Steiner, Kerstin
Hagelueken, Gregor
Messner, Paul
Schäffer, Christina
Naismith, James H.
Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus
title Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus
title_full Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus
title_fullStr Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus
title_full_unstemmed Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus
title_short Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus
title_sort structural basis of substrate binding in wsaf, a rhamnosyltransferase from geobacillus stearothermophilus
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898925/
https://www.ncbi.nlm.nih.gov/pubmed/20097205
http://dx.doi.org/10.1016/j.jmb.2010.01.035
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