High-resolution NMR spectroscopy of lipid A molecules containing 4-amino-4-deoxy-L-arabinose and phosphoethanolamine substituents -: Different attachment sites on lipid A molecules from NH4VO3-treated Escherichia coli versus kdsA mutants of Salmonella typhimurium

When Escherichia coli are grown on LB broth containing 25 mM NH4VO3, complex modifications to the lipid A anchor of lipopolysaccharide are induced. Six modified lipid As (EV1-EV6) have been purified. Many of these variants possess 4-amino-4-deoxy-L-arabinose (L-Ara4N) and/or phosphoethanolamine (pEtN) substituents, Here we use NMR spectroscopy to investigate the attachment sites of the L-Ara4N and pEtN moieties on underivatized, intact EV3 and EV6 and on precursors IIA and IIIA from kdsA mutants of Salmonella. CDCl3/CD3/OD/D2O (2:3:1, v/v) is shown to be a superior solvent for homo- and heteronuclear one- and two-dimensional NMR experiments, The latter were not feasible previously because available solvents caused sample decomposition. Selective inverse decoupling difference spectroscopy is used to determine the attachment sites of substituents on EV3, EV6, IIA, and IIIA. L-Ara4N is attached via a phosphodiester linkage to the 4'-phosphates of EV3 and EV6 and has the beta anomeric configuration. pEtN is attached by a pyrophosphate linkage to the 1-phosphate of EV6, The L-Ara4N and pEtN substituents of lipids IIA and IIIA are attached in the opposite manner, with L-Ara4N on the 1-phosphate of IIA and pEtN on the 4'-phosphate of IIIA. Determination of the proper attachment sites of these substituents is necessary for elucidating the enzymology of lipid A biosynthesis and for characterizing polymyxin-resistant mutants, in which L-Ara4N and pEtN substituents are greatly increased.