STD-NMR studies of two acceptor substrates of GlfT2, a galactofuranosyltransferase from Mycobacterium tuberculosis: epitope mapping studies.
The major structural component of the mycobacterial cell wall, the mycolyl-arabinogalactan-peptidoglycan 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)7CH3 (2) and beta-D-Galf-(1-->5)-beta-D-Galf-(1-->6)-beta-D-Galf-O(CH2)7CH3 (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.
Szczepina
Monica G
MG
Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
Zheng
Ruixiang B
RB
Completo
Gladys C
GC
Lowary
Todd L
TL
Pinto
B Mario
BM
D002240:Carbohydrate Sequence; D020134:Catalytic Domain; D005690:Galactose / Q000031:analogs & derivatives / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D013379:Substrate Specificity; D014312:Trisaccharides / Q000378:metabolism*; D014530:Uridine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism
D014312:Trisaccharides; C104136:uridine diphosphate galactofuranose; D014530:Uridine Diphosphate; D005700:Galactosyltransferases; D005690:Galactose