PMID:23536839

Citation	Simeone, R, Huet, G, Constant, P, Malaga, W, Lemassu, A, Laval, F, Daffé, M, Guilhot, C and Chalut, C (2013) Functional Characterisation of Three O-methyltransferases Involved in the Biosynthesis of Phenolglycolipids in Mycobacterium tuberculosis. PLoS ONE 8:e58954
Abstract	Phenolic glycolipids are produced by a very limited number of slow-growing mycobacterial species, most of which are pathogen for humans. In Mycobacterium tuberculosis, the etiologic agent of tuberculosis, these molecules play a role in the pathogenicity by modulating the host immune response during infection. The major variant of phenolic glycolipids produced by M. tuberculosis, named PGL-tb, consists of a large lipid core terminated by a glycosylated aromatic nucleus. The carbohydrate part is composed of three sugar residues, two rhamnosyl units and a terminal fucosyl residue, which is per-O-methylated, and seems to be important for pathogenicity. While most of the genes responsible for the synthesis of the lipid core domain and the saccharide appendage of PGL-tb have been characterized, the enzymes involved in the O-methylation of the fucosyl residue of PGL-tb remain unknown. In this study we report the identification and characterization of the methyltransferases required for the O-methylation of the terminal fucosyl residue of PGL-tb. These enzymes are encoded by genes Rv2954c, Rv2955c and Rv2956. Mutants of M. tuberculosis harboring deletion within these genes were constructed. Purification and analysis of the phenolglycolipids produced by these strains, using a combination of mass spectrometry and NMR spectroscopy, revealed that Rv2954c, Rv2955c and Rv2956 encode the methyltransferases that respectively catalysed the O-methylation of the hydroxyl groups located at positions 3, 4 and 2 of the terminal fucosyl residue of PGL-tb. Our data also suggest that methylation at these positions is a sequential process, starting with position 2, followed by positions 4 and 3.
Links	PubMed PMC3594219 Online version:10.1371/journal.pone.0058954
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
MYCTU:I6X5U4	enables	GO:0008171: O-methyltransferase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
MYCTU:I6X5U4		GO:0008171: O-methyltransferase activity	ECO:0000314:		F		Figure 4 shows that Rv2954c is responsible for the o-methylation of the hydroxyl group at position 3 of the terminal fucosyl residue of the phenolic glycolipids produced by M. tuberculosis. This conclusion is supported by the differences observed in the biochemical analyses of the glycolipid product of the Rv2954c mutant (PMM115:pPET1)compared to the same analyses of the glycolipid product of the wild-type strain. The biochemical analyses presented in figure 4 include: MALDI-TOF Mass Spec, 1D 1H-NMR, and 2D-COSY.	complete CACAO 7410
MYCTU:I6Y242		GO:0008171: O-methyltransferase activity	ECO:0000314:		F		Figure 6 shows that Rv2956 is responsible for catalyzing the O-methylation of position 2 of the terminal fucosyl residue of the phenolic glycolipid produced by M. tuberculosis H37Rv. Figure 6 shows the results of the biochemical analyses of the glycolipid products of the Rv2956 mutant strain.	complete CACAO 7651
MYCTU:I6Y242	enables	GO:0008171: O-methyltransferase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
MYCTU:P95137		GO:0008171: O-methyltransferase activity	ECO:0000314:		F		Figure 5 shows that Rv2955c is responsible for catalyzing the o-methylation of position 4 of the fucosyl residue of the phenolic glycolipid produced by M. tuberculosis H37Rv. Figure 5 shows the results of the biochemical analyses of the glycolipid products generated in the two Rv2955c mutant strains.	complete CACAO 7417
MYCTU:P95137	enables	GO:0008171: O-methyltransferase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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