Isolation, purification and properties of an intermediate in 3-deoxy-D-manno-octulosonic acid--lipid A biosynthesis

Abstract

Incomplete lipid A has been purified from a mutant of Salmonella typhimurium which is temperature-sensitive both in synthesis of 3-deoxy-D-manno-octulosonic acid 8-phosphate (dOclA-8-P) and in growth. Pulse-chase experiments have shown that the incomplete lipid A molecule is the intermediate in the biosynthesis of the dOclA-lipid A portion of lipopolysaccharides. The purification procedure included DEAE-cellulose chromatography and electrodialysis. A highly water-soluble precursor material was obtained, consisting of glucosamine, phosphate and 3-hydroxymyristic acid in a molar ratio of 1:1.2:2.1. Labeling experiments as well as chemical degradation procedures revealed the precursor molecule to be composed of a diphosphorylated glucosamine-disaccharide carrying two amide-linked and two ester-linked 3-hydroxymyristic acids. In contrast to the complete dOclA-lipid A part, the intermediate lacks 3-deoxy-D-manno-octulosonic acid as well as nonhydroxylated fatty acids. On the basis of these findings a pathway for the final steps in dOclA-lipid A biosynthesis is proposed.