dc.contributor.author | Owings, Joshua P | |
dc.contributor.author | Kuiper, Emily G | |
dc.contributor.author | Prezioso, Samantha M | |
dc.contributor.author | Meisner, Jeffrey | |
dc.contributor.author | Varga, John J | |
dc.contributor.author | Zelinskaya, Natalia | |
dc.contributor.author | Dammer, Eric B | |
dc.contributor.author | Duong, Duc M | |
dc.contributor.author | Seyfried, Nicholas T | |
dc.contributor.author | Alberti, Sebastian | |
dc.contributor.author | Conn, Graeme L | |
dc.contributor.author | Goldberg, Joanna B | |
dc.date.accessioned | 2024-07-09T09:13:08Z | |
dc.date.available | 2024-07-09T09:13:08Z | |
dc.date.issued | 2016-02-12 | |
dc.identifier.citation | Owings JP, Kuiper EG, Prezioso SM, Meisner J, Varga JJ, Zelinskaya N, et al. Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase. J Biol Chem. 2016 Feb 12;291(7):3280-90. Epub 2015 Dec 16. | en |
dc.identifier.other | http://hdl.handle.net/20.500.13003/10472 | |
dc.identifier.uri | http://hdl.handle.net/20.500.12105/20249 | |
dc.description.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 degrees C) but not at higher temperatures (37 degrees 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 degrees C abolished methyltransferase activity, whereas this activity was retained when EftM was preincubated at 25 degrees C. Irreversible protein unfolding at 37 degrees 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. | en |
dc.description.sponsorship | This work was supported in part through Cystic Fibrosis Foundation Grants GOLDBE10G0 and GOLDBE14P0 (to J. B. G.), National Institutes of Health Grant R21AI103651 (to J. B. G.), and Ministerio de Economia y Competitividad of Spain Grant SAF2012-38426 and Spanish Network for Research in Infectious Diseases Grant REIPI RD12/0015 from the Instituto de Salud Carlos III (both co-financed by the European Development Regional Fund) (to S. A.). Mass spectrometry was supported by Emory Neuroscience NINDS, National Institutes of Health, Core Facilities Grant P30NS055077. The Auto-iTC<INF>200</INF> instrument was purchased with support National Science Foundation MRI program Grant 1040177, the Winship Cancer Institute's shared resource program, and the Biochemistry Department of Emory University. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.; Supported in part by NIAID, National Institutes of Health, Training Grant T32AI007046 (to the University of Virginia).; Supported by Agriculture and Food Research Initiative Competitive Grant 2013-67011-21133 from the United States Department of Agriculture National Institute of Food and Agriculture.; Supported in part by NIAID, National Institutes of Health, Training Grant T32AI106699 (to Emory University). | es_ES |
dc.language.iso | eng | en |
dc.publisher | Amer Soc Biochemistry Molecular Biology Inc | en |
dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | * |
dc.subject | Circular dichroism (CD) | |
dc.subject | Homology modeling | |
dc.subject | Mass spectrometry (MS) | |
dc.subject | Pseudomonas aeruginosa (Paeruginosa) | |
dc.subject | S-adenosylmethionine (SAM) | |
dc.subject | Translation elongation factor | |
dc.subject | EF-Tu | |
dc.subject | Lysine methyltransferase | |
dc.subject.mesh | Bacterial Proteins | * |
dc.subject.mesh | Binding Sites | * |
dc.subject.mesh | Sequence Homology, Amino Acid | * |
dc.subject.mesh | Computational Biology | * |
dc.subject.mesh | Models, Molecular | * |
dc.subject.mesh | Isoenzymes | * |
dc.subject.mesh | Lysine | * |
dc.subject.mesh | Methylation | * |
dc.subject.mesh | Enzyme Stability | * |
dc.subject.mesh | Peptide Elongation Factor Tu | * |
dc.subject.mesh | S-Adenosylmethionine | * |
dc.subject.mesh | Protein Processing, Post-Translational | * |
dc.subject.mesh | Mutation | * |
dc.subject.mesh | Amino Acid Substitution | * |
dc.subject.mesh | Protein Methyltransferases | * |
dc.subject.mesh | Protein Unfolding | * |
dc.subject.mesh | Pseudomonas aeruginosa | * |
dc.subject.mesh | Substrate Specificity | * |
dc.subject.mesh | Protein Conformation | * |
dc.subject.mesh | Recombinant Fusion Proteins | * |
dc.subject.mesh | Temperature | * |
dc.title | Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase | en |
dc.type | research article | en |
dc.rights.license | Attribution 4.0 International | * |
dc.identifier.pubmedID | 26677219 | es_ES |
dc.format.volume | 291 | es_ES |
dc.format.number | 7 | es_ES |
dc.format.page | 3280-3290 | es_ES |
dc.identifier.doi | 10.1074/jbc.M115.706853 | |
dc.identifier.e-issn | 1083-351X | es_ES |
dc.relation.publisherversion | https://dx.doi.org/10.1074/jbc.M115.706853 | en |
dc.identifier.journal | Journal of Biological Chemistry | es_ES |
dc.rights.accessRights | open access | en |
dc.subject.decs | Sustitución de Aminoácidos | * |
dc.subject.decs | Temperatura | * |
dc.subject.decs | Especificidad por Sustrato | * |
dc.subject.decs | Mutación | * |
dc.subject.decs | Estabilidad de Enzimas | * |
dc.subject.decs | Isoenzimas | * |
dc.subject.decs | Modelos Moleculares | * |
dc.subject.decs | Desplegamiento Proteico | * |
dc.subject.decs | Sitios de Unión | * |
dc.subject.decs | S-Adenosilmetionina | * |
dc.subject.decs | Lisina | * |
dc.subject.decs | Metilación | * |
dc.subject.decs | Factor Tu de Elongación Peptídica | * |
dc.subject.decs | Procesamiento Proteico-Postraduccional | * |
dc.subject.decs | Conformación Proteica | * |
dc.subject.decs | Proteínas Recombinantes de Fusión | * |
dc.subject.decs | Biología Computacional | * |
dc.subject.decs | Proteínas Bacterianas | * |
dc.subject.decs | Proteína Metiltransferasas | * |
dc.subject.decs | Pseudomonas aeruginosa | * |
dc.subject.decs | Homología de Secuencia de Aminoácido | * |
dc.identifier.scopus | 2-s2.0-84971641403 | |
dc.identifier.wos | 370854500014 | |
dc.identifier.pui | L609983222 | |