Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase
| العنوان: | Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase |
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| المؤلفون: | Eric B. Dammer, Samantha M. Prezioso, Joanna B. Goldberg, Jeffrey Meisner, Nicholas T. Seyfried, Natalia Zelinskaya, Duc M. Duong, Sebastián Albertí, John J. Varga, Graeme L. Conn, Emily G. Kuiper, Joshua P. Owings |
| بيانات النشر: | Amer Soc Biochemistry Molecular Biology Inc, 2016. |
| سنة النشر: | 2016 |
| مصطلحات موضوعية: | 0301 basic medicine, Models, Molecular, S-Adenosylmethionine, Methyltransferase, Arginine, Protein Conformation, conformación de proteínas, metilación, homology modeling, Lysine, Sequence Homology, Biochemistry, desplegamiento proteico, especificidad del sustrato, translation elongation factor, Substrate Specificity, Enzyme Stability, lysine methyltransferase, estabilidad enzimática, chemistry.chemical_classification, biología computacional, biology, aeruginosa), Pseudomonas aeruginosa (P, Temperature, sitios de unión, sustitución de aminoácidos, Methylation, S-adenosylmethionine (SAM), Isoenzymes, proteínas bacterianas, Pseudomonas aeruginosa, proteínas de fusión recombinantes, proteína metiltransferasas, homología de secuencias, S-adenosilmetionina, EF-Tu, Recombinant Fusion Proteins, isoenzimas, Peptide Elongation Factor Tu, Microbiology, 03 medical and health sciences, Bacterial Proteins, circular dichroism (CD), mass spectrometry (MS), Protein Methyltransferases, Binding site, Thermolabile, Molecular Biology, mutación, Protein Unfolding, Binding Sites, 030102 biochemistry & molecular biology, Sequence Homology, Amino Acid, temperatura, Computational Biology, Cell Biology, factor Tu de elongación peptídica, Enzyme assay, lisina, 030104 developmental biology, Enzyme, chemistry, Amino Acid Substitution, Mutation, biology.protein, Protein Processing, Post-Translational |
| الوصف: | 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. 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-iTC200 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). |
| اللغة: | English |
| DOI: | 10.1074/jbc.m115.706853 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::2543c3405684255baa3f4e494d6f4269 https://doi.org/10.1074/jbc.m115.706853 |
| Rights: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....2543c3405684255baa3f4e494d6f4269 |
| قاعدة البيانات: | OpenAIRE |
| DOI: | 10.1074/jbc.m115.706853 |
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