High-resolution NMR Spectroscopy of Lipid A Molecules Containing 4-Amino-4-deoxy-l-arabinose and Phosphoethanolamine Substituents

Abstract

When Escherichia coli are grown on LB broth contain- ing 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 ofthe L-Ara4N and pEtN moieties on underivatized, intact EV3 and EV6 and on precursors IIA and IIIA from kdsA mutants of Salmonella. CDCl3/CD3OD/D2O (2:3:1, v/v) is shown to be a superior solvent for homo- and heteronuclear one- and two-dimensional NMR experi- ments. The latter were not feasible previously because available solvents caused sample decomposition. Selec- tive 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 phos- phodiester linkage to the 4ⴕ-phosphates of EV3 and EV6 and has the ␤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 4ⴕ-phosphate of IIIA. Determination of the proper attachment sites of these substituents is necessary for elucidating the enzy- mology of lipid A biosynthesis and for characterizing polymyxin-resistant mutants, in which L-Ara4N and pEtN substituents are greatly increased.