The major structural component of the cell wall of Mycobacterium tuberculosis is a lipidated polysaccharide, the mycoyl-arabinogalactan-peptidoglycan (mAGP) complex. This glycoconjugate plays a key role in the survival of the organism, and thus, enzymes involved in its biosynthesis have attracted attention as sites for drug action. At the core of the mAGP is a galactan composed of D-galactofuranose residues attached via alternating beta-(1-->5) and beta-(1-->6) linkages. A single enzyme, glfT, has been shown to synthesize both glycosidic linkages. We report here the first high-level expression and purification of glfT by expression of the Rv3808c gene in Escherichia coli C41(DE3). Following a three-step purification procedure, 3-7 mg of protein of >95% purity was isolated from each liter of culture. We subsequently probed the substrate specificity of glfT by evaluating a panel of potential mono- and oligosaccharide substrates and demonstrated, for the first time, that trisaccharides are better substrates than disaccharides and that one disaccharide, in which the terminal D-galactofuranose residue is replaced with an L-arabinofuranose moiety, is a weak substrate. Kinetic characterization of the enzyme using four of the oligosaccharide acceptors gave K(m) values ranging from 204 microM to 1.7 mM. Through the use of NMR spectroscopy and mass spectrometry, we demonstrated that this recombinant enzyme, like the wild-type protein, is bifunctional and can synthesize both beta-(1-->6) and beta-(1-->5)-linkages in an alternating fashion. Access to purified glfT is expected to facilitate the development of high-throughput assays for the identification of inhibitors of the enzyme, which are potential antituberculosis agents.

Two galactosyl transferases can apparently account for the full biosynthesis of the cell wall galactan of mycobacteria. Evidence is presented based on enzymatic incubations with purified natural and synthetic galactofuranose (Galf) acceptors that the recombinant galactofuranosyl transferase, GlfT1, from Mycobacterium smegmatis, the Mycobacterium tuberculosis Rv3782 ortholog known to be involved in the initial steps of galactan formation, harbors dual ␤-(134) and ␤-(135) Galf transferase activities and that the product of the enzyme, decaprenyl-P-P-GlcNAc-Rha-Galf-Galf, serves as a direct substrate for full polymerization catalyzed by another bifunctional Galf transferase, GlfT2, the Rv3808c enzyme.The mycobacterial cell wall-including the essential, covalently linked complex of peptidoglycan, heteropolymeric arabinogalactan, and highly hydrophobic mycolic acids-is responsible for many of the pathophysiological features of members of the Mycobacterium genus (9). Several antituberculosis drugs affect the formation of this complex. Isoniazid, ethionamide, thiocarlide, and thiacetazone inhibit mycolic acid synthesis (14,29,30,36,38); ethambutol specifically disrupts the synthesis of arabinan (20,24,35,39); and D-cycloserine, an inhibitor of peptidoglycan synthesis (11), has some clinical utility. However, drug resistance, particularly the multiple and extensive forms, is of pressing public health concern (15, 31), presaging the need for a broader array of targets and drugs affecting both cell wall synthesis and other aspects of mycobacterial metabolism. However, our understanding of the synthesis of the mycobacterial cell wall is elementary compared to that of other bacteria.The initiation point for arabinogalactan biogenesis is the mycobacterial version of the bacterial carrier lipid, bactoprenol, identified as decaprenyl phosphate (C 50 -P) (10), and the sequential addition of GlcNAc-P, rhamnose (Rha), and single galactofuranose (Galf) units, donated by the appropriate nucleotide sugars (23, 25) (Fig. 1). At some stage, the arabinofuranose (Araf) units are added, donated not by a nucleotide sugar but a lipid carrier, C 50 -P-Araf (20, 39). Several of the responsible glycosyl transferases taking part in this process have been identified (1,3,5,18,19,25,26,32,34).We recently described the galactofuranosyl transferase, Rv3782, responsible for attaching the first and, perhaps, the second Galf unit to the C 50 -P-P-GlcNAc-Rha acceptor (22). Due to its role in the initiation of galactan formation, we now name it galactofuranosyl transferase 1 (GlfT1). Previously, yet another galactofuranosyl transferase, Rv3808c (originally called GlfT but now named GlfT2), was recognized and proved to be bifunctional in that it was responsible for the formation of the bulk of the galactan, containing alternating 5-and 6-linked ␤-Galf units (19,25,32). In the present study, we examine the precise roles of GlfT1 and GlfT2 in mycobacterial cell wall galactan synthesis through the application of in vitro reactions with purified natural acc...