Abstract
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.
Highlights
The methylmannose polysaccharide(MMP),found in described (2). This enzyme was observedto be most active on the cytoplasm of Mycobacterium smegmatis, is com- a substrate composed of four 3-0-methylmannose units, and posed of 10 to 13 3-0-methylmannoses joined by it was successively less active with those of a larger size (2), al-4 linkages
Because MMP homologs isolated from thecell are terminated by an unmethylated mannose, the methyltransferase appears to be responsible for MMP chain termination
Acceptor Preparation-MMP, was subjected to partial methanolysis (2), and the products were fractionated by size on Bio-Gel P-4 (Fig. 1)Oligosaccharidesdiffering by only one mannose did not resolve from each other; each peak contained a mixture of both Man-OCH3were chro- (Man)-MeMan,OCH3 and MeMan,OCH
Summary
This enzyme was observedto be most active on the cytoplasm of Mycobacterium smegmatis, is com- a substrate composed of four 3-0-methylmannose units, and posed of 10 to 13 3-0-methylmannoses joined by it was successively less active with those of a larger size (2), al-4 linkages. Descending chromatography on Whatman No 1paper was performed in 1-butanol/pyridine/ water (1033, v/v). The mixture of aand pl-A-mannobiose obtained was resolved by preparative, descending chromatography on Whatman No 3MM paper that was together with the methyltransferase, which suggests eluted with ethyl acetate/propan-2-ol/water(16105,v/v) for 129 h. Assays were performed in 50 mM glycylglycine, pH 8.2, containing 10 mM MgC12. The abbreviations used are: MMP, 3-0-methylmannosepolysac- In some of the experiments, the 150,000 X g supernatant methylcharide; MeMan, 3-0-methylmannose; MMP,, the mixture of homo- transferase preparation was purified further bygel filtration. The enzyme specific activity was doubled after gel filtration whereas the blank activity was decreased by about one-half (data not shown)
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