Journal
BIOORGANIC & MEDICINAL CHEMISTRY
Volume 18, Issue 14, Pages 5123-5128Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.bmc.2010.05.069
Keywords
STD-NMR; Epitope mapping; Galactofuranosyltransferase; GlfT2; Trisaccharide substrates
Funding
- Alberta Ingenuity Centre for Carbohydrate Science
- Natural Sciences and Engineering Research Council of Canada
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The major structural component of the mycobacterial cell wall, the mycolyl-arabinogalactan-peptidogly-can complex, possesses a galactan core composed of approximately 30 galactofuranosyl (Galf) resides attached via alternating beta-(1 -> 6) and beta-(1 -> 5) linkages. Recent studies have shown that the entire galactan is synthesized by two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2. We report here saturation transfer difference (STD) NMR studies GlfT2 using two trisaccharide acceptor substrates, beta-D-Galf-(1 -> 6)-beta-D-Galf-(1 -> 5)-beta-D-Galf-O(CH2)(7)CH3 (2) and beta-D-Galf-(1 -> 5)-beta-D-Galf-(1 -> 6)-beta-D-Galf-O(CH2)(7)CH3 (3), as well as the donor substrate for the enzyme, UDP-Galf. Epitope mapping demonstrated a greater enhancement toward the 'reducing' ends of both trisaccharides, and that UDP-galactofuranose (UDP-Galf) made more intimate contacts through its nucleotide moiety. This observation is consistent with the greater flexibility required within the active site of the reaction between the growing polymer acceptor and the UDP-Galf donor. The addition of UDP-Galf to either 2 or 3 in the presence of GlfT2 generated a tetrasaccharide product, indicating that the enzyme was catalytically active. (C) 2010 Elsevier Ltd. All rights reserved.
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