title: Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation.
abstract: 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.
authors.affiliation: Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Vasileios I
authors.initials: VI
authors.lastname: Petrou
authors.affiliation: Department of Infectious Diseases, University of Georgia, College of Veterinary Medicine, Athens, GA 30602, USA.
authors.forename: Carmen M
authors.initials: CM
authors.lastname: Herrera
authors.affiliation: Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Schultz
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Oliver B
authors.initials: OB
authors.lastname: Clarke
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA. Department of Systems Biology, Columbia University, New York, NY 10032, USA.
authors.forename: Jérémie
authors.initials: J
authors.lastname: Vendome
authors.affiliation: Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: David
authors.initials: D
authors.lastname: Tomasek
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Northeastern Collaborative Access Team, Advanced Photon Source, Argonne, IL 60439, USA.
authors.forename: Surajit
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Northeastern Collaborative Access Team, Advanced Photon Source, Argonne, IL 60439, USA.
authors.forename: Kanagalaghatta R
authors.initials: KR
authors.lastname: Rajashankar
authors.affiliation: Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Meagan
authors.initials: M
authors.lastname: Belcher Dufrisne
authors.affiliation: New York Consortium on Membrane Protein Structure, New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Kloss
authors.affiliation: Department of Informatics, Bioinformatics and Computational Biology, Technische Universität München, Boltzmannstrasse 3, 85748 Garching, Germany.
authors.forename: Edda
authors.initials: E
authors.lastname: Kloppmann
authors.affiliation: Department of Informatics, Bioinformatics and Computational Biology, Technische Universität München, Boltzmannstrasse 3, 85748 Garching, Germany. Institute for Advanced Study (TUM-IAS), Technische Universität München, Boltzmannstrasse 3, 85748 Garching, Germany.
authors.forename: Burkhard
authors.initials: B
authors.lastname: Rost
authors.affiliation: Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Candice S
authors.initials: CS
authors.lastname: Klug
authors.affiliation: Department of Infectious Diseases, University of Georgia, College of Veterinary Medicine, Athens, GA 30602, USA.
authors.forename: M Stephen
authors.initials: MS
authors.lastname: Trent
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Lawrence
authors.initials: L
authors.lastname: Shapiro
authors.affiliation: Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA. fm123@cumc.columbia.edu.
authors.forename: Filippo
authors.initials: F
authors.lastname: Mancia
mesh_terms: D000606:Amino Sugars / Q000737:chemistry; D001089:Arabinose / Q000031:analogs & derivatives* / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D049921:Cupriavidus / Q000201:enzymology*; D006031:Glycosylation; D008050:Lipid A / Q000737:chemistry*; D016296:Mutagenesis; D009154:Mutation; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D011106:Polyisoprenyl Phosphates / Q000737:chemistry; D011113:Polymyxins / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D000606:Amino Sugars; D001426:Bacterial Proteins; D008050:Lipid A; D011106:Polyisoprenyl Phosphates; D011113:Polymyxins; C016982:aminoarabinose; C009621:undecaprenyl phosphate; C040134:4-amino-4-deoxyarabinose; D001089:Arabinose; D010430:Pentosyltransferases