| I. Discovery of CDP-diglyceride-dependent cytidylylation. I have found that the enzyme CDP-diglyceride hydrolase catalyzes the following reactions in vitro: (1) Transfer of the CMP moiety from CDP-diglyceride to water, phosphate, and numerous phosphomonoesters; (2) {('32)P}phosphatidic acid:CDP-diglyceride and {('32)P}inorganic phosphate:CDP exchange; and (3) dCDP-diglyceride hydrolysis and dCMP transfer.;These biochemical and genetic studies suggest a biosynthetic role for CDP-diglyceride hydrolase.;II. Identification of a novel liponucleotide involved in the biosynthesis of bacterial endotoxin. The glycolipids 2,3-diacyl-glucosamine 1-phosphate and 2,(beta)2,3-triacyl-glucosamine 1-phosphate accumulate in mutants harboring both the pgsA444 and pgsBl lesions. Using a new radiochemical method for the analysis of minor phospholipids, I have detected 2,3-diacyl-glucosamine 1-phosphate in wild-type E. coli. In addition, I have discovered a novel liponucleotide, UDP-2,3-diacyl-glucosamine, which represents 0.005% of the wild-type phospholipid and also accumulates 50- to 100-fold in pgsB mutants. Pulse-labeling experiments indicate that UDP-2,3-diacyl-glucosamine is synthesized prior to 2,3-diacyl-glucosamine 1-phosphate. These data suggest that the following two reactions are steps in lipid A biosynthesis. (1) UDP-2,3-diacyl-glucosamine (--->) 2,3-diacyl-glucosamine 1-phosphate + UMP; (2) UDP-2,3-diacyl-glucosamine + 2,3-diacyl-glucosamine 1-phosphate (--->) 2,3,2',3'-tetraacyl-disaccharide 1-phosphate.;Lastly, I have identified a membrane-bound enzyme which converts, 2,3-diacyl-glucosamine 1-phosphate to 2,(beta)2,3-triacyl-glucosamine 1-phosphate using an endogenous acyl donor.;I have isolated mutants defective in CDP-diglyceride hydrolase. This genetic locus, designated cdh, maps at minute 88, appears to be the structural gene, and is nonessential for growth. Hydrolase mutants are defective in both hydrolysis and cytidylylation, and accumulate CDP- and dCDP-diglyceride in vivo. |
