PMID:6370995

Ray, BL, Painter, G and Raetz, CR (1984) The biosynthesis of gram-negative endotoxin. Formation of lipid A disaccharides from monosaccharide precursors in extracts of Escherichia coli.J. Biol. Chem. 259:4852-9

Abstract

We have discovered an enzyme in the cytosol of Escherichia coli that generates lipid A disaccharides from monosaccharide precursors by the following route: 2,3-diacyl-GlcN-1-P + UDP-2,3-diacyl-GlcN---- 2,3-diacyl-GlcN (beta, 1----6) 2,3-diacyl-GlcN-1-P + UDP. Previous studies from our laboratory have documented the presence in vivo of the precursors 2,3-diacylglucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) (lipid X of E. coli) and UDP-2,3-diacylglucosamine (UDP-2,3-diacyl-GlcN) (Bulawa, C.E., and Raetz, C.R.H.J. Biol. Chem. 259, 4846-4851). Both substrates are novel glucosamine-derived phospholipids, acylated with beta-hydroxymyristoyl moieties, and they accumulate in E. coli mutants defective in the pgsB gene. Synthetic ADP-, GDP-, and CDP-2,3-diacylglucosamines are inefficient substrates compared to the naturally occurring UDP derivative. The free-acid form of the tetraacyldisaccharide 1-phosphate product (C68H129N2O20P) that is generated in vitro has Mr = 1325.74 as judged by fast atom bombardment mass spectrometry. Mild acid hydrolysis (0.1 M HCl for 30 min at 100 degrees C) liberates greater than 95% of the phosphate moiety as Pi. Detailed analysis by 1H and 13C NMR spectroscopy confirms the presence of a phosphate residue at position 1 of the disaccharide, an alpha-anomeric configuration at the reducing end, and a beta, 1----6 linkage between the two glucosamines. Importantly the disaccharide 1-phosphate synthase is missing in extracts of E. coli strains harboring the pgsB1 mutation, consistent with the massive accumulation of 2,3-diacyl-GlcN-1-P and UDP-2,3-diacyl-GlcN in vivo. The enzymatic reaction reported here represents a major biosynthetic route for the formation of lipid A disaccharides in E. coli and other Gram-negative bacteria. An in vitro system for the biosynthesis of lipid A disaccharides has not been described previously.

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Annotations

Gene product	Qualifier	GO ID	GO term name	Evidence Code	with/from	Aspect	Notes	Status
LpxB		GO:0008915	lipid-A-disaccharide synthase activity	IDA: Inferred from Direct Assay		F	The conversion of UDP-2,3diacylglucosamine and [32P]2,3diacylglucosamine-1-phosphate into [32P]2,3-diacylglucosamine-(beta-1,6)2,3-diacylglucosamine-1-phosphate (lipid A disaccharide or tetraacyldisaccharide-1-phosphate) is monitored by thin layer chromatography and scintillation counting (Fig. 3). Product formation can also be followed using a differential extraction procedure (Fig. 4) obviating the need for the chromatographic step.	complete
LpxB		GO:0009245	lipid A biosynthetic process	IMP: Inferred from Mutant Phenotype		P	Extracts prepared from a strain with the pgsB1 (psgB has been renamed lpxB) mutation are markedly deficient in their ability to synthesize the lipid A disaccharide from its precursors (Fig. 9).	complete
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