Galactan biosynthesis in Mycobacterium tuberculosis. Identification of a bifunctional UDP-galactofuranosyltransferase

• Published in: The Journal of biological chemistry 2001-07, Vol.276 (28), p.26430-26440
• Main Authors: Kremer, L, Dover, L G, Morehouse, C, Hitchin, P, Everett, M, Morris, H R, Dell, A, Brennan, P J, McNeil, M R, Flaherty, C, Duncan, K, Besra, G S
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - metabolism; galactans; Galactans - biosynthesis; Galactosyltransferases - metabolism; Mycobacterium tuberculosis; Mycobacterium tuberculosis - metabolism; Substrate Specificity; UDPgalactofuranosyltransferase
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M102022200

The cell wall of Mycobacterium tuberculosis and related genera is unique among prokaryotes, consisting of a covalently bound complex of mycolic acids, D-arabinan and D-galactan, which is linked to peptidoglycan via a special linkage unit consisting of Rhap-(1-->3)-GlcNAc-P. Information concerning the biosynthesis of this entire polymer is now emerging with the promise of new drug targets against tuberculosis. Accordingly, we have developed a galactosyltransferase assay that utilizes the disaccharide neoglycolipid acceptors beta-d-Galf-(1-->5)-beta-D-Galf-O-C(10:1) and beta-D-Galf-(1-->6)-beta-D-Galf-O-C(10:1), with UDP-Gal in conjunction with isolated membranes. Chemical analysis of the subsequent reaction products established that the enzymatically synthesized products contained both beta-D-Galf linkages ((1-->5) and (1-->6)) found within the mycobacterial cell, as well as in an alternating (1-->5) and (1-->6) fashion consistent with the established structure of the cell wall. Furthermore, through a detailed examination of the M. tuberculosis genome, we have shown that the gene product of Rv3808c, now termed glfT, is a novel UDP-galactofuranosyltransferase. This enzyme possesses dual functionality in performing both (1-->5) and (1-->6) galactofuranosyltransferase reactions with the above neoglycolipid acceptors, using membranes isolated from the heterologous host Escherichia coli expressing Rv3808c. Thus, at a biochemical and genetic level, the polymerization of the galactan region of the mycolyl-arabinogalactan complex has been defined, allowing the possibility of further studies toward substrate recognition and catalysis and assay development. Ultimately, this may also lead to a more rational approach to drug design to be explored in the context of mycobacterial infections.