Biosynthesis of the mycobacterial methylmannose polysaccharide. Identification of a 3-O-methyltransferase.

The methylmannose polysaccharide (MMP), found in the cytoplasm of Mycobacterium smegmatis, is composed of 10 to 13 3-O-methylmannoses joined by alpha 1----4 linkages. Each molecule is terminated by an unmethylated mannose and the reducing end is blocked by an alpha-linked methyl aglycon. Two enzymes involved in MMP biosynthesis have been identified in cell extracts, an alpha 1----4 mannosyltransferase (described in the previous paper) and a 3-O-methyltransferase reported here. Studies of substrate specificity and characterization of the products formed demonstrate that MMP elongation occurs via sequential mannosylation and methylation. The 3-O-methyltransferase, unlike the mannosyltransferase, is readily solubilized. It catalyzes transfer of a methyl group from S-adenosylmethionine to position 3 of a terminal alpha 1----4-linked mannose. The labeled product formed from S-[methyl-3H]adenosylmethionine and Man-MeMan5-OCH3 was characterized both by its resistance to periodate oxidation and by the release of labeled 3-O-methylmannose upon acid hydrolysis. Like the mannosyltransferase, the 3-O-methyltransferase utilizes shorter oligomeric acceptors preferentially. The Km values of the methyltransferase for Man-MeMan4-OCH3 and S-adenosylmethionine are 0.7 and 0.4 mM, respectively. Because MMP homologs isolated from the cell are terminated by an unmethylated mannose, the methyl-transferase appears to be responsible for MMP chain termination. Moreover, palmitoyl-CoA selectively inhibits methylation of Man-MeMan12-OCH3 when Man-MeMan4-OCH3 and Man-MeMan12-OCH3 are incubated together with the methyltransferase, which suggests that complex formation between the longer homologs and lipids may play a role in the termination process.