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

Abstract

The lipid A disaccharide of the Escherichia coli envelope is synthesized from the two fatty acylated glucosamine derivatives UDP-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., & 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., & Raetz, C. R. H. (1985) J. Biol. Chem. 260, 15536-15541; Anderson, M. S., & 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-N2,O3- bis[(R)-3-hydroxytetradecanoyl]-beta-D-glucopyranosyl]-(1----6)-2-amino- 2-deoxy-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D- glucopyranose 1-phosphate (tetra-acyldisaccharide-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 other 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.

Biosynthesis of a structurally novel lipid A in Rhizobium leguminosarum: identification and characterization of six metabolic steps leading from UDP-GlcNAc to 3-deoxy-D-manno-2-octulosonic acid2-lipid IVA

“…[a-P] UDPGlcNAc was prepared and incubated with unlabeled R-3-hydroxymyristoyl-ACP. As noted earlier (3,14,22), upon the addition of crude cytosolic protein from E. coli, the labeled substrate was rapidly metabolized to mono-and diacylated species, as judged by TLC (Fig. 3).…”

“…Other 3-hydroxy fatty acids are 3-hydroxymyristic acid (3-OH-C14:0), 3-hydroxypentadecanoic acid (3-OH-C15o0), 3-hydroxypalmitic acid (3-OH-C16o0), and 3-hydroxystearic acid (3-OH-C18:0) (6). The type and quantity of these acyl groups vary among different Rhizobium species.…”

Lipid A Modification Systems in Gram-Negative Bacteria

“…Thus, the deacetylation of UDP-3-O-(acyl)-GlcNAc by LpxC is the actual committed step of Kdo 2 -lipid A biosynthesis (74,97). LpxC is a Zn 2+ -dependent enzyme that is highly conserved in all gram-negative bacteria (98,99).…”

Structural basis for the acyl chain selectivity and mechanism of UDP- N -acetylglucosamine acyltransferase

“…1) (8). Because LpxA catalyzes a thermodynamically unfavorable reaction (K eq Ϸ 0.01) (8), the second enzyme of the pathway, the deacetylase (LpxC), is the committed step (9,10). LpxA and LpxC are essential for growth (10)(11)(12), and both are validated targets for the design of novel antibiotics (13)(14)(15).…”