Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 287, Issue 33, Pages 28132-28143Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M112.347484
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Funding
- Alberta Glycomics Centre
- Natural Sciences and Engineering Research Council
- Alberta Heritage Foundation for Medical Research Senior Scholar Award
- Department of Energy Office of Biological and Environmental Research
- National Institutes of Health from NIGMS [P41GM103393]
- NCRR [P41RR001209]
- Natural Sciences and Engineering Research Council of Canada
- National Research Council Canada
- Canadian Institutes of Health Research
- Province of Saskatchewan
- Western Economic Diversification Canada
- University of Saskatchewan
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Biosynthesis of the mycobacterial cell wall relies on the activities of many enzymes, including several glycosyltransferases (GTs). The polymerizing galactofuranosyltransferase GlfT2 (Rv3808c) synthesizes the bulk of the galactan portion of the mycolyl-arabinogalactan complex, which is the largest component of the mycobacterial cell wall. We used x-ray crystallography to determine the 2.45-angstrom resolution crystal structure of GlfT2, revealing an unprecedented multidomain structure in which an N-terminal beta-barrel domain and two primarily alpha-helical C-terminal domains flank a central GT-A domain. The kidney- shaped protomers assemble into a C-4-symmetric homotetramer with an open central core and a surface containing exposed hydrophobic and positively charged residues likely involved with membrane binding. The structure of a 3.1-angstrom resolution complex of GlfT2 withUDPreveals a distinctive mode of nucleotide recognition. In addition, models for the binding of UDP-galactofuranose and acceptor substrates in combination with site-directed mutagenesis and kinetic studies suggest a mechanism that explains the unique ability of GlfT2 to generate alternating beta-(1 -> 5) and beta-(1 -> 6) glycosidic linkages using a single active site. The topology imposed by docking a tetrameric assembly onto a membrane bilayer also provides novel insights into aspects of processivity and chain length regulation in this and possibly other polymerizing GTs.
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