Biosynthesis of a Mycobacterial Lipopolysaccharide

Abstract

Abstract A particulate enzyme preparation was obtained from Mycobacterium phlei cells which had the activity of a polysaccharide:acyl coenzyme A acyltransferse. We conclude that the enzyme system is involved in the biosynthesis of the methylglucose-containing lipopolysaccharide (MGLP), since it catalyzed the transfer of acetyl, propionyl, isobutyryl, octanoyl, and succinyl groups, all of which are known to be present in the lipopolysaccharide. Moreover, the enzyme preparation used α-(1→4)-d-glucooligosaccharides as acceptors, a result consistent with the fact that a major part of the polysaccharide component of the lipopolysaccharide has the same amylose-like structure. Both MGLP and its deacylated product, MGP, acted as acceptors of acyl groups, but MGP which had been partially acetylated by acetic anhydride was still more active. The random acetylation probably increased slightly the hydrophobic character of MGP and promoted its binding to the enzyme. In agreement with this idea was the fact that artificial d-glucooligosaccharide acceptors were completely inactive unless partially acetylated. Previous studies have established that five of the eight positions of acylation in MGLP-III are located on the four sugar units at the nonreducing end of the polysaccharide. From analysis of the product obtained in the enzymic reaction between [14C]acetyl-CoA and MGP, we now have evidence that at least two of the sites for acetylation occur in this tetrasaccharide unit. Removal of the four sugar units from MGP by the combined action of α-amylase and glucoamylase I destroyed the capacity of the polysaccharide to accept acetyl groups from acetyl-CoA. However, this partially degraded MGP retained acceptor activity for succinyl groups, which suggests that the sites for succinylation are located elsewhere in the polysaccharide, a result which is in agreement with direct chemical analysis of the lipopolysaccharide (Smith, W. L., and Ballou, C. E. (1973) J. Biol. Chem. 248, 7118–7125).