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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.

The methylmannose polysaccharide, found in the cytoplasm of Mycobacterium smegmatis, is composed of 3-O-methylmannose units joined in alpha 1----4 linkage in a chain terminated by unmethylated mannose at the nonreducing end. An alpha 1----4-mannosyltransferase, one of the two enzymes involved in methylmannose polysaccharide elongation, has been identified in cell extracts. The activity is membrane-associated and catalyzes the transfer of mannose from GDP-mannose to oligomeric acceptors composed of 4 to 12 3-O-methylmannoses. 1H NMR spectroscopy and alpha-mannosidase digestion confirm that the mannose is attached by an alpha 1----4 linkage. In competition studies, the enzyme utilizes shorter oligomeric acceptors preferentially. The Km of the mannosyltransferase for MeMan4-OCH3 is 15-20 microM, for MeMan6-OCH3 it is 75-85 microM, and for GDP-mannose it is 55 microM.

The longitudinal nuclear magnetic relaxation times of the methyl protons of alpha-methyl-D-mannopyranoside have been measured at 90 MHz and 270 MHz in solutions of concanavalin A complexed with: (i) Mn2+ and Ca2+; and (ii) Zn2+ and Ca2+. Zn2+ and Mn2+ are known to bind in site S1 and Ca2+ in site S2 of concanavalin A. Both sites must be occupied before monosaccharides will bind to the protein. In order to extract T1p, the paramagnetic contribution to the bound methyl relaxation time, from these observations the relaxation time of uncomplexed sugar was determined. Free Mn2+ contributed insignificantly to the latter value; however, outer sphere relaxation was found to be large at bound Mn2+ concentrations greater than 1 mM. Comparison of the results obtained at the two frequencies allowed the determination of the correlation time for the interaction between the methyl protons and the bound Mn2+ and the distance between them (21.5 +/- 1.2 A). In contrast to previous results from nuclear magnetic resonance studies, this distance is consistent with binding at the cavity proposed for the saccharide binding site by Becker et al. [J. Biol. Chem. 250, 1513 (1975)], although it does not preclude possible binding sites on the surface of the molecule.

Vicia faba lectin contained 40-57% beta-conformation, 4-23% alpha-conformation along with random coil at pH 7.2 depending upon the analytical methods used. The percentage of beta-conformation increased with the addition of N-acetyl-D-glucosamine or methyl alpha-D-mannopyranoside. The structural transitions of V. faba lectin were affected by alkali at pH 9.6 and 10.6. Binding constants and free energy changes for the interaction between V. faba lectin and N-acetyl-D-glucosamine and methyl alpha-D-mannopyranoside were estimated at pH 7.2 using the c.d. and fluorescence methods.