Biosynthesis of the Linkage Region of the Mycobacterial Cell Wall

Abstract

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 [14C]GlcNAc from UDP-[14C]GlcNAc into two glycolipids (1 and 2) and of [14C]Rha from TDP-[14C]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-[14C]Galp and a preparation of cell walls allowed the incorporation of [14C]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.

Biosynthesis of Mycobacterial Lipoarabinomannan

“…However, shortly after this initial work, Takayama et al (36,37) and Schultz and Elbein (38) described two alkali-stable mannophospholipids in M. tuberculosis and M. smegmatis, a mannosyl-1-phosphoryl-decaprenol (C 50 -P-Man) and a mannosyl-1-phosphoryl heptaprenol (C 35 -P-Man), which, in light of their group transfer potential and known role in mannolipid synthesis from other organisms (39), could be donors of polymerized Man in mycobacterial cell walls. Indeed, Schultz and co-workers (38,40) 14 C]Gal, and [ 14 C]Ara from their corresponding donors into the mycobacterial cell wall "core" (15,18). Incorporation in all cases was quantitatively and qualitatively comparable.…”

The nucleoside diphosphate kinase of Mycobacterium smegmatis: identification of proteins that modulate specificity of nucleoside triphosphate synthesis by the enzyme

“…Very little is known about the details of the biosynthesis of arabinomannans or other polysaccharides that might use CDP-or UDP-linked sugars as intermediates and may therefore require a large pool of CTP or UTP. Indeed, the incorporation of UDP-[ 14 C]-GlcNAc and UDP-[ 14 C]-Gal in various mycobacterial glycolipid components has recently been demonstrated (Mikusova et al, 1996). The availability of the antibodies against the M. smegmatis proteins will allow us to look for the presence of similar proteins in the virulent strains of M. tuberculosis or even Mycobacterium leprae by Western blotting and determine their intracellular levels at various growth phases.…”

Novel prenyl-linked benzophenone substrate analogues of mycobacterial mannosyltransferases

“…In addition, decaprenyl diphosphate N-acetylglucosamine is involved in the biosynthesis of peptidoglycan and the initial stages of the 'linker unit' synthesis for mAGP in M. tuberculosis [39]. Clearly, enzymes that utilize polyprenyl phosphate carriers, whether for mycolylation or polysaccharide synthesis, are attractive targets for drug development due to the essentiality of PIM/LM and the mAGP complex and their subsequent identification key to future chemotherapeutic strategies [1,38].…”