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

Abstract

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.

Tetrameric Structure of the GlfT2 Galactofuranosyltransferase Reveals a Scaffold for the Assembly of Mycobacterial Arabinogalactan

“…5, A and B). This mechanism is consistent with the mutagenesis results described above, the presence of a single GT-A domain, and a single active site per GT protomer, in addition to saturation-transfer difference-NMR studies (13).…”

“…GlfT2 has been more extensively studied than GlfT1. Of particular note are saturation-transfer difference-NMR studies suggesting that GlfT2 synthesizes both the ␤-(135) and ␤-(136) linkages using a single active site (13), and mass spectrometry studies indicate that the enzyme is processive (14). A very recent study examining GlfT2 mutants provides additional support for the presence of a single active site that carries out both glycosylations (15).…”

The Mycobacterial Cell Wall—Peptidoglycan and Arabinogalactan

“…Apart from being a bifunctional GalT, GlfT2 displays additional interesting features: By using a substrate tethering mechanism, it is able to have intrinsic control of the chain length of the galactan product (May et al 2009). Further structural analysis of GlfT2 using X-ray crystallography and NMR has shed further light on how this unique enzyme is tetrameric and is able to control chain length processivity and bifunctionality using a single active site (Szczepina et al 2009;Wheatley et al 2012). …”

NMR Spectroscopy for Studying Interactions of Bioactive Molecules

“…The results of competitive STD-NMR experiments indicated that erythromycin A binds to the active site and acts as a competitive glycosidase inhibitor. Similarly, STD-NMR spectroscopy was used to study the interaction between bifunctional galactofuranosyltranferase GlfT2 and two trisaccharide acceptor substrates [61]. The bifunctionality of the enzyme was explored by competition STD experiments and saturation transfer double difference experiments (STDD).…”