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 moiety.
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 alpha-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 beta, 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 beta-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 degrees 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.
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Department of Biochemistry and the Duke NMR Spectroscopy Center and Department of Radiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Ribeiro
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Raetz
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D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D005942:Gluconates / Q000032:analysis* / Q000378:metabolism; D005944:Glucosamine / Q000032:analysis; D006090:Gram-Negative Bacteria / Q000737:chemistry; D006885:Hydroxybutyrates / Q000032:analysis* / Q000378:metabolism; D008050:Lipid A / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D012231:Rhizobium / Q000737:chemistry* / Q000502:physiology
D005942:Gluconates; D006885:Hydroxybutyrates; D008050:Lipid A; D005944:Glucosamine