Biosynthesis of lipid A precursors in Escherichia coli. A cytoplasmic acyltransferase that converts UDP-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine.

Preliminary studies from our laboratory have suggested the existence of a novel set of fatty acyltransferases in extracts of Escherichia coli that attach two R-3-hydroxymyristoyl moieties to UDP-GlcNAc (Anderson, M.S., Bulawa, C.E., and Raetz, C.R.H. (1985) J. Biol. Chem. 260, 15536-15541). The resulting "glucosamine-derived" phospholipids appear to be crucial precursors for the biosynthesis of the lipid A component of lipopolysaccharide. We now describe an assay and a 1000-fold purification of the first enzyme in this pathway, which catalyzes the reaction: UDP-GlcNAc + R-3-hydroxymyristoyl-acyl carrier protein----UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc + acyl carrier protein. The covalent structure of the monoacylated UDP-GlcNAc product was established by fast atom bombardment mass spectrometry and 1H-NMR spectroscopy. The UDP-GlcNAc acyltransferase has a strict requirement for R-3-hydroxymyristoyl-acyl carrier protein, since R-3-hydroxymyristoyl coenzyme A and myristoyl-acyl carrier protein are not substrates. Of various NDP-GlcNAc preparations examined, only the uridine and thymidine derivatives were utilized to a significant extent. When the product of the reaction (UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc) was isolated and reincubated with crude E. coli extracts, it was rapidly converted to more hydrophobic products in the presence of R-3-hydroxymyristoyl-acyl carrier protein. We propose that the addition of an R-3-hydroxymyristoyl residue to the 3 position of the GlcNAc moiety of UDP-GlcNAc is the first committed step in lipid A biosynthesis and that UDP-GlcNAc is situated at a biosynthetic branchpoint in E. coli leading either to lipid A or to peptidoglycan.