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, comple x modifications to the lipid A anchor of lipopolysaccharide are induced. Si x modified lipid As (EV1-EV6) have been purified. Many of these variants po ssess 4-amino-4-deoxy-L-arabinose (L-Ara4N) and/or phosphoethanolamine (pEt N) substituents, Here we use NMR spectroscopy to investigate the attachment sites of the L-Ara4N and pEtN moieties on underivatized, intact EV3 and EV 6 and on precursors II, and III, from kdsA mutants of Salmonella. CDCl3/CD3 /OD/D2O (2:3:1, v/v) is shown to be a superior solvent for homo- and hetero nuclear one- and two-dimensional NMR experiments, The latter were not feasi ble previously because available solvents caused sample decomposition. Sele ctive inverse decoupling difference spectroscopy is used to determine the a ttachment sites of substituents on EV3, EV6, IIA, and IIIA. L-Ara4N is atta ched via a phosphodiester linkage to the 4'-phosphates of EV3 and EV6 and h as the beta anomeric configuration. pEtN is attached by a pyrophosphate lin kage 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-ph osphate of IIA and pEtN on the 4'-phosphate of IIIA. Determination of the p roper attachment sites of these substituents is necessary for elucidating t he enzymology of lipid A biosynthesis and for characterizing polymyxin-resi stant mutants, in which L-Ara4N and pEtN substituents are greatly increased .