Polymerization of Mycobacterial Arabinogalactan and Ligation to Peptidoglycan

Tetsuya Yagi,Sebabrata Mahapatra,Katarína Mikušová,Dean C Crick,Patrick J Brennan

doi:10.1074/jbc.m302216200

Tetsuya Yagi, Sebabrata Mahapatra + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m302216200

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Abstract

The cell wall of Mycobacterium spp. consists predominately of arabinogalactan chains linked at the reducing ends to peptidoglycan via a P-GlcNAc-(alpha1-3)-Rha linkage unit (LU) and esterified to a variety of mycolic acids at the nonreducing ends. Several aspects of the biosynthesis of this complex have been defined, including the initial formation of the LU on a polyprenyl phosphate (Pol-P) molecule followed by the sequential addition of galactofuranosyl (Galf) units to generate Pol-P-P-LU-(Galf)1,2,3, etc. and Pol-P-P-LU-galactan, catalyzed by a bifunctional galactosyltransferase (Rv3808c) capable of adding alternating 5- and 6-linked Galf units. By applying cell-free extracts of Mycobacterium smegmatis, containing cell wall and membrane fragments, and differential labeling with UDP-[14C]Galp and recombinant UDP-Galp mutase as the source of [14C]Galf for galactan biosynthesis and 5-P-[14C]ribosyl-P-P as a donor of [14C]Araf for arabinan synthesis, we now demonstrate sequential synthesis of the simpler Pol-P-P-LU-(Galf)n glycolipid intermediates followed by the Pol-P-P-LU-arabinogalactan and, finally, ligation of the P-LU-arabinogalactan to peptidoglycan. This first time demonstration of in vitro ligation of newly synthesized P-LU-arabinogalactan to newly synthesized peptidoglycan is a necessary forerunner to defining the genetics and enzymology of cell wall polymer-peptidoglycan ligation in Mycobacterium spp. and examining this step as a target for new antibacterial drugs.

Highlights

Mycolic acids interspersed with a variety of free lipids, lipoglycans, and proteins [1]; there is evidence for polysaccharides on the outer face of the cell wall [2]
By applying cell-free extracts of Mycobacterium smegmatis, containing cell wall and membrane fragments, and differential labeling with UDP-[14C]Galp and recombinant UDP-Galp mutase as the source of [14C]Galf for galactan biosynthesis and 5-P-[14C]ribosyl-P-P as a donor of [14C]Araf for arabinan synthesis, we demonstrate sequential synthesis of the simpler polyprenyl phosphate (Pol-P)-P-linkage unit (LU)-(Galf)n glycolipid intermediates followed by the Pol-P-P-LU-arabinogalactan and, ligation of the P-LU-arabinogalactan to peptidoglycan
The mycolic acids are attached to the nonreducing ends of the arabinogalactan, whereas the reducing ends are covalently attached to the crosslinked peptidoglycan via phosphoryl-N-acetylglucosaminosylrhamnosyl linkage units (P-GlcNAc-Rha)

Summary

Polymerization of Mycobacterial Arabinogalactan and Ligation to Peptidoglycan*

By applying cell-free extracts of Mycobacterium smegmatis, containing cell wall and membrane fragments, and differential labeling with UDP-[14C]Galp and recombinant UDP-Galp mutase as the source of [14C]Galf for galactan biosynthesis and 5-P-[14C]ribosyl-P-P as a donor of [14C]Araf for arabinan synthesis, we demonstrate sequential synthesis of the simpler Pol-P-P-LU-(Galf)n glycolipid intermediates followed by the Pol-P-P-LU-arabinogalactan and, ligation of the P-LU-arabinogalactan to peptidoglycan. This first time demonstration of in vitro ligation of newly synthesized P-LUarabinogalactan to newly synthesized peptidoglycan is a necessary forerunner to defining the genetics and enzymology of cell wall polymer-peptidoglycan ligation in Mycobacterium spp. and examining this step as a target for new antibacterial drugs. § Present address: Dept. of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina CH-1, Bratislava 842 15, Slovakia

EXPERIMENTAL PROCEDURES

RESULTS

Insoluble residue

DISCUSSION