Please use this identifier to cite or link to this item:http://hdl.handle.net/20.500.12105/20249
Title
Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase
Author(s)
Date issued
2016-02-12
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.
Language
Inglés
Document type
research article
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.
Subject
Circular dichroism (CD) | Homology modeling | Mass spectrometry (MS) | Pseudomonas aeruginosa (Paeruginosa) | S-adenosylmethionine (SAM) | Translation elongation factor | EF-Tu | Lysine methyltransferase
MESH
Bacterial Proteins | Binding Sites | Sequence Homology, Amino Acid | Computational Biology | Models, Molecular | Isoenzymes | Lysine | Methylation | Enzyme Stability | Peptide Elongation Factor Tu | S-Adenosylmethionine | Protein Processing, Post-Translational | Mutation | Amino Acid Substitution | Protein Methyltransferases | Protein Unfolding | Pseudomonas aeruginosa | Substrate Specificity | Protein Conformation | Recombinant Fusion Proteins | Temperature
DECS
Sustitución de Aminoácidos | Temperatura | Especificidad por Sustrato | Mutación | Estabilidad de Enzimas | Isoenzimas | Modelos Moleculares | Desplegamiento Proteico | Sitios de Unión | S-Adenosilmetionina | Lisina | Metilación | Factor Tu de Elongación Peptídica | Procesamiento Proteico-Postraduccional | Conformación Proteica | Proteínas Recombinantes de Fusión | Biología Computacional | Proteínas Bacterianas | Proteína Metiltransferasas | Pseudomonas aeruginosa | Homología de Secuencia de Aminoácido
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