Biosynthesis of a Mycobacterial Lipopolysaccharide: EVIDENCE FOR AN ACYLPOLYSACCHARIDE METHYLTRANSFERASE

Abstract

Abstract It was suggested recently that a soluble methyltransferase from Mycobacterium phlei, which was shown to transfer methyl groups from S-adenosylmethionine to position 6 of α(1 → 4)-linked d-glucooligosaccharides, may function in the biosynthesis of the 10 α(1 → 4)-linked 6-O-methyl-d-glucose residues of a mycobacterial lipopolysaccharide (Ferguson, J. A., and Ballou, C. E. (1970) J. Biol. Chem. 245, 4213). In a reinvestigation of this system, we have failed to observe methylation by the M. phlei extract unless the glucooligosaccharides were partially acetylated. We have studied this phenomenon further and have determined some of the characteristics of the reaction. Different oligosaccharide preparations with 0.2 to 1.6 acetyl groups per glucose unit were active as acceptors. At low levels of acetylation (0.2 acetyls per glucose), methylation occurred only at position 6. At high levels of acetylation (1.6 acetyls per glucose), methylation at position 6 was strongly depressed, and some methylation then occurred at position 3 as well. The 6-O-methyltransferase was studied in some detail by the use of oligosaccharides with low levels of acetylation. Partially acetylated oligosaccharides, with 5 to 15 glucose residues, were readily methylated. However, only a trace of methylation was obtained with the tetrasaccharide, and no activity was observed with the di- or trisaccharide, or with cyclooctaamylose. Digestion of the enzymically methylated octasaccharide with β-amylase gave products which suggested that methylation had occurred only at sites which were more than 4 glucose residues from the nonreducing end. This is consistent with the fact that the 6-O-methylglucose-containing section of the lipopolysaccharide itself begins 4 glucose units in from the nonreducing end. The structural similarity between the methylated octasaccharide and the mycobacterial lipopolysaccharide suggests that the 6-O-methyltransferase is specific for the methylation of a partially acylated or otherwise lipophilic precursor of the lipopolysaccharide.