STD-NMR Studies Suggest that Two Acceptor Substrates for GlfT2, a Bifunctional Galactofuranosyltransferase Required for the Biosynthesis of Mycobacterium tuberculosis Arabinogalactan, Compete for the Same Binding Site

• Published in: Chembiochem : a European journal of chemical biology 2009-08, Vol.10 (12), p.2052-2059
• Main Authors: Szczepina, Monica G, Zheng, Ruixiang Blake, Completo, Gladys C, Lowary, Todd L, Pinto, B. Mario
• Format: Article
• Language: English
• Subjects: Binding Sites; enzymes; Galactans - biosynthesis; Galactosyltransferases - metabolism; glycosylation; mycobacteria; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - metabolism; NMR spectroscopy; Nuclear Magnetic Resonance, Biomolecular; oligosaccharides
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.200900202

The mycobacterial cell wall is a complex architecture, which has, as its major structural component, a lipidated polysaccharide covalently bound to peptidoglycan. This structure, termed the mycolyl-arabinogalactan-peptidoglycan complex, possesses a core galactan moiety composed of approximately 30 galactofuranosyl (Galf) resides attached via alternating β-(1[rightward arrow]6) and β-(1[rightward arrow]5) linkages. Recent studies have shown that the entire galactan is synthesized by the action of only two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2. We report here saturation-transfer difference (STD) NMR spectroscopy studies with GlfT2 using two trisaccharide acceptor substrates, β-D-Galf-(1[rightward arrow]6)-β-D-Galf-(1[rightward arrow]5)-β-D-Galf-O(CH₂)₇CH₃ (2) and β-D-Galf-(1[rightward arrow]5)-β-D-Galf-(1[rightward arrow]6)-β-D-Galf-O(CH₂)₇CH₃ (3), as well as the donor substrate for the enzyme, UDP-Galf. Competition STD-NMR titration experiments and saturation transfer double difference (STDD) experiments with 2 and 3 were undertaken to explore the bifunctionality of this enzyme, in particular to answer whether one or two active sites are responsible for the formation of both β-(1[rightward arrow]5)- and β-(1[rightward arrow]6)-Galf linkages. It was demonstrated that 2 and 3 bind competitively at the same site; this suggests that GlfT2 has one active site pocket capable of catalyzing both β-(1[rightward arrow]5) and β-(1[rightward arrow]6) galactofuranosyl transfer reactions. The addition of UDP-Galf to GlfT2 in the presence of either 2 or 3 generated a tetrasaccharide product; this indicates that the enzyme was catalytically active under the conditions at which the STD-NMR experiments were carried out.