title: ArnT proteins that catalyze the glycosylation of lipopolysaccharide share common features with bacterial N-oligosaccharyltransferases.
abstract: ArnT is a glycosyltransferase that catalyzes the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to the lipid A moiety of the lipopolysaccharide. This is a critical modification enabling bacteria to resist killing by antimicrobial peptides. ArnT is an integral inner membrane protein consisting of 13 predicted transmembrane helices and a large periplasmic C-terminal domain. We report here the identification of a functional motif with a canonical consensus sequence DEXRYAX(5)MX(3)GXWX(9)YFEKPX(4)W spanning the first periplasmic loop, which is highly conserved in all ArnT proteins examined. Site-directed mutagenesis demonstrated the contribution of this motif in ArnT function, suggesting that these proteins have a common mechanism. We also demonstrate that the Burkholderia cenocepacia and Salmonella enterica serovar Typhimurium ArnT C-terminal domain is required for polymyxin B resistance in vivo. Deletion of the C-terminal domain in B. cenocepacia ArnT resulted in a protein with significantly reduced in vitro binding to a lipid A fluorescent substrate and unable to catalyze lipid A modification with l-Ara4N. An in silico predicted structural model of ArnT strongly resembled the tertiary structure of Campylobacter lari PglB, a bacterial oligosaccharyltransferase involved in protein N-glycosylation. Therefore, distantly related oligosaccharyltransferases from ArnT and PglB families operating on lipid and polypeptide substrates, respectively, share unexpected structural similarity that could not be predicted from direct amino acid sequence comparisons. We propose that lipid A and protein glycosylation enzymes share a conserved catalytic mechanism despite their evolutionary divergence.
authors.affiliation: Department of Microbiology and Immunology, University of Western Ontario, London, ON, Canada N6A 5C1 Instituto de Biotecnología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León. Nuevo León, Mexico.
authors.forename: Faviola
authors.initials: F
authors.lastname: Tavares-Carreón
authors.affiliation: Centre for Infection and Immunity, Queen's University Belfast, Belfast BT9 5GZ, UK Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt.
authors.forename: Yasmine
authors.initials: Y
authors.lastname: Fathy Mohamed
authors.affiliation: Department of Microbiology and Immunology, University of Western Ontario, London, ON, Canada N6A 5C1 Departamento de Microbiología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico.
authors.forename: Angel
authors.initials: A
authors.lastname: Andrade
authors.affiliation: Department of Microbiology and Immunology, University of Western Ontario, London, ON, Canada N6A 5C1 Centre for Infection and Immunity, Queen's University Belfast, Belfast BT9 5GZ, UK m.valvano@qub.ac.uk.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Valvano
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000606:Amino Sugars / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001089:Arabinose / Q000737:chemistry / Q000378:metabolism; D057508:Burkholderia cenocepacia / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008050:Lipid A / Q000737:chemistry / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019779:Salmonella enterica / Q000201:enzymology
chemical_list: D000606:Amino Sugars; D008050:Lipid A; D008070:Lipopolysaccharides; D008565:Membrane Proteins; C040134:4-amino-4-deoxyarabinose; D001089:Arabinose; D006602:Hexosyltransferases; C442226:L-Ara4N transferase
keywords: antimicrobial peptides; lipid A modification; membrane proteins; oligosaccharyltransferases