Biosynthesis of the linkage region of the mycobacterial cell wall

The ''core'' structure of the cell wall of Mycobacterium and related genera is unique among prokaryotes, consisting of a covalently linked complex of mycolic acids, D-arabinan and D-galactan (mycolylarabinogalactan, mAG), which, in turn, is linked to peptidoglycan via a special linkage unit, -alpha-L-Rhap(1-->3)-D-GlcNAc-P-. Little is known of the biosynthesis of this complex, although it is the site of action of several common anti-tuberculosis drugs. Isolated cell membranes of Mycobacterium smegmatis catalyzed the incorporation of [C-14]GlcNAc from UDP-[C-14]GlcNAc into two glycolipids (1 and 2) and of [C-14]Rha from TDP-[C-14]Rha into glycolipid 2. These products were characterized as polyprenol-P-P-GlcNAc (glycolipid 1) and polyprenol-P-P-GlcNAc-Rha (glycolipid 2) based on sensitivity of synthesis to tunicamycin, chromatographic characterization of the products of mild acid hydrolysis, and mass spectral analysis of the glycosyl and polyprenyl units. Glycolipids 1 and 2 were shown to be precursors of the linkage unit in polymerized cell wall. The inclusion in the assays of UDP-[C-14]Galp and a preparation of cell walls allowed the incorporation of [C-14]Gal into two further glycolipids (3 and 4). Preliminary evidence indicates a precursor-product relationship among glycolipids 1, 2, 3, and 4. Thus, the first steps in the biosynthesis of the mycobacterial cell wall involve synthesis of the linkage disaccharide on a polyprenyl-P-P carrier followed by growth of the galactan unit. Assays are thus defined for the screening of new anti-tuberculosis drugs active against cell wall synthesis.