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oai:repisalud.isciii.es:20.500.12105/202492024-09-22T04:50:58Zcom_20.500.12105_15322com_20.500.12105_2051col_20.500.12105_16967

00925njm 22002777a 4500 dc Owings, Joshua P author Kuiper, Emily G author Prezioso, Samantha M author Meisner, Jeffrey author Varga, John J author Zelinskaya, Natalia author Dammer, Eric B author Duong, Duc M author Seyfried, Nicholas T author Alberti, Sebastian author Conn, Graeme L author Goldberg, Joanna B author 2016-02-12 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. 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. 10.1074/jbc.M115.706853 1083-351X Journal of Biological Chemistry http://hdl.handle.net/20.500.13003/10472 26677219 L609983222 2-s2.0-84971641403 http://hdl.handle.net/20.500.12105/20249 370854500014 Circular dichroism (CD) Homology modeling Mass spectrometry (MS) Pseudomonas aeruginosa (Paeruginosa) S-adenosylmethionine (SAM) Translation elongation factor EF-Tu Lysine methyltransferase Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase