In vitro synthesis of a lipid-linked trisaccharide involved in synthesis of enterobacterial common antigen

Journal Article · Wed Mar 01 04:00:00 EST 1989 · J. Bacteriol.; (United States)

OSTI ID:6327187

Barr, K; Nunes-Edwards, P; Rick, P D

The heteropolysaccharide chains of enterobacterial common antigen (ECA) are made up of linear trisaccharide repeat units with the structure----3)-alpha-D-Fuc4NAc-(1----4)- beta-D-ManNAcA-(1----4)-alpha-D-GlcNAc-(1----, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. The assembly of these chains involves lipid-linked intermediates, and both GlcNAc-pyrophosphorylundecaprenol (lipid I) and ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) are intermediates in ECA biosynthesis. In this study we demonstrated that lipid II serves as the acceptor of Fuc4NAc residues in the assembly of the trisaccharide repeat unit of ECA chains. Incubation of Escherichia coli membranes with UDP-GlcNAc, UDP-(14C)ManNAcA, and TDP-(3H)Fuc4NAc resulted in the synthesis of a radioactive glycolipid (lipid III) that contained both (14C)ManNAcA and (3H)Fuc4NAc. The oligosaccharide moiety of lipid III was identified as a trisaccharide by gel-permeation chromatography, and the in vitro synthesis of lipid III was dependent on prior synthesis of lipids I and II. Accordingly, the incorporation of (3H)Fuc4NAc into lipid III from the donor TDP-(3H)Fuc4NAc was dependent on the presence of both UDP-GlcNAc and UDP-ManNAcA in the reaction mixtures. In addition, the in vitro synthesis of lipid III was abolished by tunicamycin. Direct conversion of lipid II to lipid III was demonstrated in two-stage reactions in which membranes were initially incubated with UDP-GlcNAc and UDP-(14C)ManNAcA to allow the synthesis of radioactive lipid II.

• Research Organization: Uniformed Services Univ. of the Health Sciences, Bethesda, MD (USA)
• OSTI ID: 6327187
• Journal Information: J. Bacteriol.; (United States), Journal Name: J. Bacteriol.; (United States) Vol. 171:3; ISSN JOBAA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANTIGENS
BACTERIA
BIOSYNTHESIS
CARBOHYDRATES
CARBON 14 COMPOUNDS
CELL CONSTITUENTS
CELL MEMBRANES
CHROMATOGRAPHY
ESCHERICHIA COLI
IN VITRO
ISOTOPE APPLICATIONS
ISOTOPE DILUTION
LABELLED COMPOUNDS
LIPIDS
MEMBRANES
MICROORGANISMS
MOLECULAR STRUCTURE
OLIGOSACCHARIDES
ORGANIC COMPOUNDS
POLYSACCHARIDES
SACCHARIDES
SALMONELLA
SALMONELLA TYPHIMURIUM
SEPARATION PROCESSES
SYNTHESIS
TRACER TECHNIQUES
TRITIUM COMPOUNDS