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PMID:6370995

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Citation

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.

Links

PubMed

Keywords

Disaccharides/biosynthesis; Endotoxins/biosynthesis; Escherichia coli/metabolism; Glucosephosphates/metabolism; Glycolipids/metabolism; Kinetics; Lipid A/biosynthesis; Magnetic Resonance Spectroscopy; Mass Spectrometry; Models, Molecular; Molecular Conformation; Phosphorus Radioisotopes/diagnostic use; Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives; Uridine Diphosphate N-Acetylglucosamine/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:LPXB

located_in

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

ECOLI:LPXB

acts_upstream_of_or_within

GO:0009245: lipid A biosynthetic process

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

ECOLI:LPXB

part_of

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

ECOLI:LPXB

part_of

GO:0005829: cytosol

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

ECOLI:LPXB

enables

GO:0008915: lipid-A-disaccharide synthase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:LPXB

involved_in

GO:0009245: lipid A biosynthetic process

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete


See also

References

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