aminoarabinose and 4-amino-4-deoxyarabinose

Polymyxins are antibiotics used in the last line of defense to combat multidrug-resistant infections by Gram-negative bacteria. Polymyxin resistance arises through charge modification of the bacterial outer membrane with the attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose to lipid A, a reaction catalyzed by the integral membrane lipid-to-lipid glycosyltransferase 4-amino-4-deoxy-L-arabinose transferase (ArnT). Here, we report crystal structures of ArnT from Cupriavidus metallidurans, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.8 and 3.2 angstrom resolution, respectively. The structures show cavities for both lipidic substrates, which converge at the active site. A structural rearrangement occurs on undecaprenyl phosphate binding, which stabilizes the active site and likely allows lipid A binding. Functional mutagenesis experiments based on these structures suggest a mechanistic model for ArnT family enzymes.

Topics: Amino Sugars; Arabinose; Bacterial Proteins; Catalysis; Catalytic Domain; Crystallography, X-Ray; Cupriavidus; Glycosylation; Lipid A; Mutagenesis; Mutation; Pentosyltransferases; Polyisoprenyl Phosphates; Polymyxins; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Substrate Specificity

Lipopolysaccharides (LPS) of two polymyxin-resistant (pmr) mutants and the corresponding parent strain of Escherichia coli were chemically analysed for composition and subjected to 31P-NMR (nuclear magnetic resonance) for assessment of phosphate substitution. Whereas the saccharide portions, fatty acids, and phosphate contents were similar in wild-type and pmr LPS, the latter contained two- to threefold higher amounts of 2-aminoethanol. The pmr LPS also contained 4-amino-4-deoxy-L-arabinopyranose (L-Arap4N), which is normally not a component of E. coli LPS. This aminopentose has been assigned to be linked to the 4'-phosphate of lipid A. Comparative 31P-NMR analysis of the de-O-acylated LPS of the wild-type and pmr strains revealed that phosphate groups of the pmr LPS were mainly (71-79%) diphosphate diesters, which accounted for only 20% in the wild-type LPS. Diphosphate monoesters were virtually nonexistent in the pmr LPS, whereas they accounted for 42% of all phosphates in wild-type LPS. In the lipid A of the pmr strains, the 4'-phosphate was to a significant degree (35%) substituted by L-Arap4N, whereas in the wild-type LPS the L-ArapN was absent. In the pmr lipid A, 2-aminoethanol was completely substituting the glycosidic pyrophosphate but not the glycosidic monophosphate, forming a diphosphate diester linkage at this position in 40% of lipid A molecules. In the wild-type LPS the glycosidic position of lipid A carried mostly unsubstituted monophosphate and pyrophosphate. Thus the polymyxin resistance was shown to be associated, along with the esterification of the lipid A 4'-monophosphate by aminoarabinose, with extensive esterification of diphosphates in LPS by 2-aminoethanol.

Topics: Amino Sugars; Arabinose; Diphosphates; Drug Resistance, Microbial; Escherichia coli; Ethanolamine; Ethanolamines; Lipid A; Lipopolysaccharides; Magnetic Resonance Spectroscopy; Mutation; Phosphates; Polymyxin B