title: 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.
authors.affiliation: Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
authors.forename: M S
authors.initials: MS
authors.lastname: Anderson
authors.forename: A D
authors.initials: AD
authors.lastname: Robertson
authors.forename: I
authors.initials: I
authors.lastname: Macher
authors.forename: C R
authors.initials: CR
authors.lastname: Raetz
mesh_terms: D000089:Acetic Anhydrides; D000213:Acyl Carrier Protein / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D007202:Indicators and Reagents; D008050:Lipid A / Q000096:biosynthesis*; D009682:Magnetic Resonance Spectroscopy; D009227:Myristic Acids / Q000138:chemical synthesis / Q000378:metabolism*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
chemical_list: D000089:Acetic Anhydrides; D000213:Acyl Carrier Protein; D007202:Indicators and Reagents; D008050:Lipid A; D009227:Myristic Acids; D014539:Uridine Diphosphate Sugars; C052121:UDP-3-O-(3-hydroxymyristoyl)-N-acetylglucosamine; C031800:acetic anhydride; D014537:Uridine Diphosphate N-Acetylglucosamine; D000255:Adenosine Triphosphate; C041030:UDP-N(2),3-di(3-hydroxymyristoyl)glucosamine