lipid-a and undecaprenyl-phosphate

The biosynthesis of glycoconjugates is remarkably conserved in all types of cells since the biochemical reactions involved exhibit similar characteristics, which can be summarized as follows: (a) the saccharide moiety is formed as a lipid-linked, membrane-associated glycan; (b) the lipid component in most cases is a polyisoprenoid phosphate; (c) the assembly of the lipid-linked saccharide intermediate depends on reactions taking place at both sides of the cell membrane, which requires the obligatory transmembrane movement of amphipathic molecules across the lipid bilayer. These general characteristics are present in the biosynthesis of the O-antigen component of the bacterial lipopolysaccharide, which serves as a model system to investigate the molecular and mechanistic basis of glycoconjugate synthesis, as summarized in this mini-review.

Topics: Bacterial Proteins; Cell Membrane; Escherichia coli Proteins; Glycoconjugates; Glycosyltransferases; Hexosyltransferases; Lipid A; Lipopolysaccharides; Membrane Lipids; Membrane Transport Proteins; O Antigens; Polyisoprenyl Phosphates; Transferases; Transferases (Other Substituted Phosphate Groups)

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

Lipid A of Francisella tularensis subsp. novicida contains a galactosamine (GalN) residue linked to its 1-phosphate group. As shown in the preceding paper, this GalN unit is transferred to lipid A from the precursor undecaprenyl phosphate-beta-D-GalN. A small portion of the free lipid A of Francisella novicida is further modified with a glucose residue at position-6'. We now demonstrate that the two F. novicida homologues of Escherichia coli ArnC, designated FlmF1 and FlmF2, are essential for lipid A modification with glucose and GalN, respectively. Recombinant FlmF1 expressed in E. coli selectively condenses undecaprenyl phosphate and UDP-glucose in vitro to form undecaprenyl phosphate-glucose. Recombinant FlmF2 selectively catalyzes the condensation of undecaprenyl phosphate and UDP-N-acetylgalactosamine to generate undecaprenyl phosphate-N-acetylgalactosamine. On the basis of an analysis of the lipid A composition of flmF1 and flmF2 mutants of F. novicida, we conclude that FlmF1 generates the donor substrate for the modification of F. novicida free lipid A with glucose, whereas FlmF2 generates the immediate precursor of the GalN donor substrate, undecaprenyl phosphate-beta-D-GalN. A novel deacetylase, present in membranes of F. novicida, removes the acetyl group from undecaprenyl phosphate-N-acetylgalactosamine to yield undecaprenyl phosphate-beta-D-GalN. This deacetylase may have an analogous function to the deformylase that generates undecaprenyl phosphate-4-amino-4-deoxy-alpha-l-arabinose from undecaprenyl phosphate-4-deoxy-4-formylamino-alpha-l-arabinose in polymyxin-resistant strains of E. coli and Salmonella typhimurium.

Topics: Acetylgalactosamine; Acetyltransferases; Bacterial Proteins; Cell Membrane; Chromatography, Liquid; Escherichia coli; Francisella; Galactosamine; Glucosides; Lipid A; Multigene Family; Mutation; Polyisoprenyl Phosphates; Recombinant Proteins; Spectrometry, Mass, Electrospray Ionization

One-third of the lipid A found in the Escherichia coli outer membrane contains an unsubstituted diphosphate unit at position 1 (lipid A 1-diphosphate). We now report an inner membrane enzyme, LpxT (YeiU), which specifically transfers a phosphate group to lipid A, forming the 1-diphosphate species. (32)P-labelled lipid A obtained from lpxT mutants do not produce lipid A 1-diphosphate. In vitro assays with Kdo(2)-[4'-(32)P]lipid A as the acceptor shows that LpxT uses undecaprenyl pyrophosphate as the substrate donor. Inhibition of lipid A 1-diphosphate formation in wild-type bacteria was demonstrated by sequestering undecaprenyl pyrophosphate with the cyclic polypeptide antibiotic bacitracin, providing evidence that undecaprenyl pyrophosphate serves as the donor substrate within whole bacteria. LpxT-catalysed phosphorylation is dependent upon transport of lipid A across the inner membrane by MsbA, a lipid A flippase, indicating a periplasmic active site. In conclusion, we demonstrate a novel pathway in the periplasmic modification of lipid A that is directly linked to the synthesis of undecaprenyl phosphate, an essential carrier lipid required for the synthesis of various bacterial polymers, such as peptidoglycan.

Topics: Anti-Bacterial Agents; ATP-Binding Cassette Transporters; Bacitracin; Bacterial Proteins; Escherichia coli K12; Lipid A; Membrane Lipids; Mutation; Peptidyl Transferases; Periplasm; Phosphates; Phosphorylation; Polyisoprenyl Phosphates; Pyrophosphatases

The recycling of the lipid carrier undecaprenyl-phosphate (Und-P) requires the dephosphorylation of Und-PP, a reaction proposed to occur at the external or periplasmic side of the bacterial cell membrane. In this issue of Molecular Microbiology, experiments based on the analysis of lipopolysaccharide modifications in Escherichia coli demonstrate that the phosphorylation of lipid A at position 1 is catalysed by the membrane enzyme LpxT (formerly YeiU). This enzyme specifically transfers the distal phosphate group from Und-PP to lipid A 1-phosphate to produce lipid A 1-diphosphate. Furthermore, this reaction requires a functionally intact MsbA protein, which catalyses the transfer of lipid A across the membrane, confirming that the LpxT-mediated lipid A modification occurs on the periplasmic side of the membrane. These observations provide a novel and unexpected link between periplasmic lipid A modifications and the Und-PP recycling pathway.

Topics: Escherichia coli; Lipid A; Models, Biological; Phosphorylation; Polyisoprenyl Phosphates; Polysaccharides