Structural characterization by (13)C-NMR spectroscopy of products synthesized in vitro by polysaccharide synthases using (13)C-enriched glycosyl donors: application to a UDP-glucose:(1-->3)-beta-D-glucan synthase from blackberry (Rubus fruticosus).
A simple and sensitive method for the characterization of products synthesized in vitro by polysaccharide synthases is described. It relies on the use of (13)C-enriched nucleotide sugars as substrates and on the analysis of the newly synthesized polysaccharides by (13)C-nuclear magnetic resonance (NMR) spectroscopy. The method was validated with a (1-->3)-beta-d-glucan synthase from blackberry, but it may be applied to the study of any glycosyltransferase. The chemical synthesis of UDP-d-[U-(13)C]glucose was achieved in a classical procedure with an overall yield of 50%. A uniformly labeled (1-->3)-beta-d-glucan was synthesized from this substrate, using detergent extracts of blackberry cell membranes as a source of synthase. One hundred micrograms of product was sufficient for liquid and solid-state (13)C-NMR spectroscopy analyses. The method is at least 100 times more sensitive than in the case of nonenriched polysaccharides. It allows the unequivocal identification and direct structural characterization of the products synthesized in vitro, as opposed to conventional methods that rely on the use of radioactive substrates and enzymatic hydrolysis of the polysaccharides with specific glycoside hydrolases. The method proves that the glycan analyzed was synthesized de novo because the final product is enriched in (13)C. Information on the 3D organization of the polymer may also be obtained by solid-state NMR spectroscopy.
Fairweather
Jon K
JK
Centre de Recherches sur les Macromolécules Végétales, affiliated with the Joseph Fourier University of Grenoble, B.P. 53, 38041 Grenoble cedex 9, France.
Him
Joséphine Lai Kee
JL
Heux
Laurent
L
Driguez
Hugues
H
Bulone
Vincent
V
D002250:Carbon Radioisotopes; D005964:Glucosyltransferases / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D027824:Rosaceae / Q000737:chemistry*; D014532:Uridine Diphosphate Glucose / Q000737:chemistry*
D002250:Carbon Radioisotopes; D005964:Glucosyltransferases; C040232:glucan synthase; D014532:Uridine Diphosphate Glucose