title: Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase.
abstract: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that trimethylates elongation factor-thermo-unstable (EF-Tu) on lysine 5. Lysine 5 methylation occurs in a temperature-dependent manner and is generally only seen when P. aeruginosa is grown at temperatures close to ambient (25 °C) but not at higher temperatures (37 °C). We have previously identified the gene, eftM (for EF-Tu-modifying enzyme), responsible for this modification and shown its activity to be associated with increased bacterial adhesion to and invasion of respiratory epithelial cells. Bioinformatic analyses predicted EftM to be a Class I S-adenosyl-l-methionine (SAM)-dependent methyltransferase. An in vitro methyltransferase assay was employed to show that, in the presence of SAM, EftM directly trimethylates EF-Tu. A natural variant of EftM, with a glycine to arginine substitution at position 50 in the predicted SAM-binding domain, lacks both SAM binding and enzyme activity. Mass spectrometry analysis of the in vitro methyltransferase reaction products revealed that EftM exclusively methylates at lysine 5 of EF-Tu in a distributive manner. Consistent with the in vivo temperature dependence of methylation of EF-Tu, preincubation of EftM at 37 °C abolished methyltransferase activity, whereas this activity was retained when EftM was preincubated at 25 °C. Irreversible protein unfolding at 37 °C was observed, and we propose that this instability is the molecular basis for the temperature dependence of EftM activity. Collectively, our results show that EftM is a thermolabile, SAM-dependent methyltransferase that directly trimethylates lysine 5 of EF-Tu in P. aeruginosa.
authors.affiliation: From the Department of Pediatrics, Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis and Sleep and the Emory-Children's Center for Cystic Fibrosis Research, Emory University and Children's Healthcare of Atlanta, Atlanta, Georgia 30322, the Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Joshua P
authors.initials: JP
authors.lastname: Owings
authors.affiliation: the Department of Biochemistry and the Biochemistry, Cell, and Developmental Biology Program and.
authors.forename: Emily G
authors.initials: EG
authors.lastname: Kuiper
authors.affiliation: From the Department of Pediatrics, Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis and Sleep and the Emory-Children's Center for Cystic Fibrosis Research, Emory University and Children's Healthcare of Atlanta, Atlanta, Georgia 30322, Microbiology and Molecular Genetics Program, Graduate Division of Biological and Biomedical Sciences, Emory University School of Medicine, Atlanta, Georgia 30322, and.
authors.forename: Samantha M
authors.initials: SM
authors.lastname: Prezioso
authors.affiliation: From the Department of Pediatrics, Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis and Sleep and the Emory-Children's Center for Cystic Fibrosis Research, Emory University and Children's Healthcare of Atlanta, Atlanta, Georgia 30322.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Meisner
authors.affiliation: From the Department of Pediatrics, Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis and Sleep and the Emory-Children's Center for Cystic Fibrosis Research, Emory University and Children's Healthcare of Atlanta, Atlanta, Georgia 30322, the Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: John J
authors.initials: JJ
authors.lastname: Varga
authors.affiliation: the Department of Biochemistry and.
authors.forename: Natalia
authors.initials: N
authors.lastname: Zelinskaya
authors.affiliation: the Department of Biochemistry and.
authors.forename: Eric B
authors.initials: EB
authors.lastname: Dammer
authors.affiliation: the Department of Biochemistry and.
authors.forename: Duc M
authors.initials: DM
authors.lastname: Duong
authors.affiliation: the Department of Biochemistry and.
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Seyfried
authors.affiliation: the Instituto Universitario de Investigación en Ciencias de la Salud, Universidad de las Islas Baleares, Palma de Mallorca, 07122 Spain.
authors.forename: Sebastián
authors.initials: S
authors.lastname: Albertí
authors.affiliation: the Department of Biochemistry and.
authors.forename: Graeme L
authors.initials: GL
authors.lastname: Conn
authors.affiliation: From the Department of Pediatrics, Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis and Sleep and the Emory-Children's Center for Cystic Fibrosis Research, Emory University and Children's Healthcare of Atlanta, Atlanta, Georgia 30322, the Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, Virginia 22908, joanna.goldberg@emory.edu.
authors.forename: Joanna B
authors.initials: JB
authors.lastname: Goldberg
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D019295:Computational Biology; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D008958:Models, Molecular; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011496:Protein Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D058767:Protein Unfolding; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000254:growth & development; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D012436:S-Adenosylmethionine; D011496:Protein Methyltransferases; D010444:Peptide Elongation Factor Tu; D008239:Lysine
keywords: EF-Tu; Pseudomonas aeruginosa (P. aeruginosa); S-adenosylmethionine (SAM); circular dichroism (CD); homology modeling; lysine methyltransferase; mass spectrometry (MS); translation elongation factor