Lipid A mutants of Salmonella typhimurium. Purification and characterization of a lipid A precursor produced by a mutant in 3-deoxy-D-mannooctulosonate-8-phosphate synthetase.
We describe here the isolation, purification, and structural characterization of a lipid A precursor synthesized under nonpermissive conditions by a mutant of Salmonella typhimurium conditionally defective in the synthesis of the 3-deoxy-D-mannoctulosonate (2-keto-3-deoxyoctonate, KDO) region of the lipopolysaccharide. The precursor was isolated free from lipopolysaccharide, murein, and phospholipids by extraction of delipidated cells with 90% phenol/CHCL3/petroleum ether. The molecule was recovered from the phenol phase after precipitation of lipopolysaccharide with H2O and subsequently purified by DEAE-cellulose chromatography. Structural analyses showed that the lipid A precursor is a phosphorylated glucosamine disaccharide containing one ester and two amide-linked residues of beta-hydroxymyristate. In contrast to lipid A, the precursor disaccharide lacks ester-linked 12:0 and 14:0 fatty acids as well as KDO. The molecule contains 2 phosphate residues both of which were identified as phosphomonoesters by 31P NMR spectroscopy. One of the phosphomonoesters is located in position 1 of the reducing terminal glucosamine residue; the location of the other phosphomonoester was not determined. The structure of the precursor provides strong support for the conclusion that KDO incorporation occurs at an early stage in lipid A biosynthesis prior to the incorporation of ester-linked saturated fatty acids.
D000446:Aldehyde-Lyases / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D004187:Disaccharides / Q000096:biosynthesis / Q000302:isolation & purification; D006017:Glycolipids / Q000096:biosynthesis / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008050:Lipid A / Q000096:biosynthesis*; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D009154:Mutation; D010428:Pentosephosphates; D010728:Phosphoenolpyruvate; D012486:Salmonella typhimurium / Q000378:metabolism*; D013400:Sugar Acids
D004187:Disaccharides; D006017:Glycolipids; D008050:Lipid A; D008070:Lipopolysaccharides; D010428:Pentosephosphates; D013400:Sugar Acids; D010728:Phosphoenolpyruvate; D000446:Aldehyde-Lyases