Isolation, Purification and Properties of an Intermediate in 3-Deoxy-d-manno-octulosonic Acid-Lipid A Biosynthesis

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Abstract
Incomplete lipid A was purified from a mutant of Salmonella typhimurium which is temperature-sensitive in the synthesis of 3-deoxy-D-manno-octulosonic acid 8-phosphate (dOc1A-8-P) and in growth. Pulse-chase experiments showed that the incomplete lipid A molecule is an intermediate in the biosynthesis of the dOc1A-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 and chemical degradation procedures showed that the precursor molecule is composed of a diphosphorylated glucosamine-disaccharide carrying 2 amide-linked and 2 ester-linked 3-hydroxymyristic acids. Unlike the complete dOc1A-lipid A part, the intermediate lacks 3-deoxy-D-manno-octulosonic acid and nonhydroxylated fatty acids. A pathway for the final steps in dOc1A-lipid A biosynthesis is proposed.