Biosynthesis of lipid A in Escherichia coli: identification of UDP-3-O-[(R)-3-hydroxymyristoyl]-.alpha.-D-glucosamine as a precursor of UDP-N2,O3-bis[(R)-3-hydroxymyristoyl]-.alpha.-D-glucosamine

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Abstract
The lipid A disaccharide of the Escherichia coli envelope is synthesized from the two fatty acylated glucosamine derivatives UPD-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-.alpha.-D-glucosamine (UDP-2,3-diacyl-GlcN) and N2,O3-bis[(R)-3-hydroxytetradecanoyl]-.alpha.-D-glucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) [Ray, B. L. Painter, G., and Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859]. We have previously shown that UDP-2,3-diacyl-GlcN is generated in extracts of E. coli by fatty acylation of UDP-GlcNAc, giving UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc as the first intermediate, which is rapidly converted to UDP-2,3-diacyl-GlcN [Anderson, M. S., Bulawa, C. E., and Raetz, C. R. H. (1985) J. Biol. Chem. 260, 15536-15541; Anderson, M. S., and Raetz, C. R. H. (1987) J. Biol. Chem. 262, 5159-5169]. We now demonstrate a novel enzyme in the cytoplasmic fraction of E. coli, capable of deacetylating UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc to form UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine. The covalent structure of the previously undescribed UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine intermediate was established by 1H NMR spectroscopy and fast atom bombardment mass spectrometry. This material can be made to accumulate in E. coli extracts upon incubation of UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc in the absence of the fatty acyl donor [(R)-3-hydroxymyristoyl]acyl carrier protein. However, addition of the isolated deacetylation product [UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine] back to membrane-free extracts of E. coli in the presence of [(R)-3-hydroxymyristoyl]-acyl carrier protein results in rapid conversion of this compound into the more hydrophobic products UDP-2,3-diacyl-GlcN, 2,3-diacyl-GlcN-1-P, and O-[2-amino-2-deoxy-N2O3-bis[(R)-3-hydroxtetradecanoyl]-.beta.-D-glucopyranosyl]-(1 .fwdarw. 6)-2-amino-2-deoxy-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-.alpha.-D-glucopyranose 1-phosphate (tetraacyldisaccharide-1-P), demonstrating its competency as a precursor. In vitro incubations using [acetyl-3H]UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc confirmed release of the acetyl moiety in this system as acetate, not as some acetyl derivative. The deacetylation reaction was inhibited by 1 mM N-ethylmaleimide, while the subsequent N-acylation reaction was not. Our observations provide strong evidence that UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine is a true intermediate in the biosynthesis of UDP-2,3-diacyl-GlcN and lipid A.

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