Two-dimensional NMR Spectroscopy and Structures of Six Lipid A Species from Rhizobium etli CE3 DETECTION OF AN ACYLOXYACYL RESIDUE IN EACH COMPONENT AND ORIGIN OF THE AMINOGLUCONATE MOIETYS

The chemical structures of six lipid A species (A, B, C, D-1, D-2, and E) purified from Rhizobium etli CE3 were investigated by one- and two-dimensional NMR spectroscopy. The R. etli lipid A subtypes each contain an unusual acyloxyacyl residue at position 2′ as part of a conserved distal glucosamine moiety but differ in their proximal units. All R. etli lipid A species lack phosphate groups. However, they are derivatized with an α -linked galacturonic acid group at position 4′, as shown by nuclear Overhauser effect spectroscopy. Component B, which had been not been reported in previous studies, features a β , 1′-6 linked disaccharide of glucosamine acylated at positions 2, 3, 2′, and 3′ in a pattern that is typical of lipid A found in other Gram-negative bacteria. D-1 contains an acylated aminogluconate unit in place of the proximal glucosamine residue of B. C and E lack ester-linked β -hydroxyacyl chains at position 3, as judged by their H-3 chemical shifts, and may be synthesized from B and D-1, respectively, by the R. etli 3- O -deacylase. D-2 is an isomer of D-1 that forms nonenzymatically by acyl chain migration. A may be an elimination product derived from D-1 during hydrolysis at 100 °C (pH 4.5), a step needed to release lipid A from lipopolysaccharide. Based on these findings, we propose a biosynthetic scheme for R. etli lipid A in which B is generated first by a variation of the E. coli pathway. The aminogluconate unit of D-1 could then be made from B by enzymatic oxidation of the proximal glucosamine. As predicted by our hypothesis, enzyme(s) can be demonstrated in extracts of R. etli that convert 14 C-labeled B to D-1.