title: Burkholderia cenocepacia and Salmonella enterica ArnT proteins that transfer 4-amino-4-deoxy-l-arabinose to lipopolysaccharide share membrane topology and functional amino acids.
abstract: We recently demonstrated that incorporation of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to the lipid A moiety of lipopolysaccharide (LPS) is required for transport of LPS to the outer membrane and viability of the Gram-negative bacterium Burkholderia cenocepacia. ArnT is a membrane protein catalyzing the transfer of l-Ara4N to the LPS molecule at the periplasmic face of the inner membrane, but its topology and mechanism of action are not well characterized. Here, we elucidate the topology of ArnT and identify key amino acids that likely contribute to its enzymatic function. PEGylation assays using a cysteineless version of ArnT support a model of 13 transmembrane helices and a large C-terminal region exposed to the periplasm. The same topological configuration is proposed for the Salmonella enterica serovar Typhimurium ArnT. Four highly conserved periplasmic residues in B. cenocepacia ArnT, tyrosine-43, lysine-69, arginine-254 and glutamic acid-493, were required for activity. Tyrosine-43 and lysine-69 span two highly conserved motifs, (42)RYA(44) and (66)YFEKP(70), that are found in ArnT homologues from other species. The same residues in S. enterica ArnT are also needed for function. We propose these aromatic and charged amino acids participate in either undecaprenyl phosphate-l-Ara4N substrate recognition or transfer of l-Ara4N to the LPS.
authors.affiliation: Centre for Human Immunology, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, N6A 5C1, Canada.
authors.forename: Faviola
authors.initials: F
authors.lastname: Tavares-Carreón
authors.affiliation: Centre for Infection and Immunity, Queen's University Belfast, Belfast, BT9 7AE, United Kingdom.
authors.forename: Kinnari B
authors.initials: KB
authors.lastname: Patel
authors.affiliation: 1] Centre for Human Immunology, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, N6A 5C1, Canada [2] Centre for Infection and Immunity, Queen's University Belfast, Belfast, BT9 7AE, United Kingdom.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Valvano
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000606:Amino Sugars / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D057508:Burkholderia cenocepacia / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D008070:Lipopolysaccharides / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D054730:Protein Interaction Domains and Motifs; D019779:Salmonella enterica / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000596:Amino Acids; D000606:Amino Sugars; D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D008565:Membrane Proteins; C040134:4-amino-4-deoxyarabinose