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.

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 beta-(1-->6) and beta-(1-->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, beta-D-Galf-(1-->6)-beta-D-Galf-(1-->5)-beta-D-Galf-O(CH(2))(7)CH(3) (2) and beta-D-Galf-(1-->5)-beta-D-Galf-(1-->6)-beta-D-Galf-O(CH(2))(7)CH(3) (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 beta-(1-->5)- and beta-(1-->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 beta-(1-->5) and beta-(1-->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.