Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase

• Type of Material: Article
• Authors: Joshua P. Owings, Emory University; Emily G. Kuiper, Emory University; Samantha M. Prezioso, Emory University; Jeffrey Meisner, Emory University; John J. Varga, Emory University; Natalia Zelinskaya, Emory University; Eric Dammer, Emory University; Duc M. Duong, Emory University; Nicholas Seyfried, Emory University; Sebastián Albertí, Universidad de las Islas Baleares; Graeme Conn, Emory University; Joanna Goldberg, Emory University
• Language: English
• Date: 2016-02-12
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1074/jbc.M115.706853
• Title of Journal or Parent Work: Journal of Biological Chemistry
• ISSN: 0021-9258
• Volume: 291
• Issue: 7
• Start Page: 3280
• End Page: 3290
• Grant/Funding Information: 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.; Mass spectrometry was supported by Emory Neuroscience NINDS, National Institutes of Health, Core Facilities Grant P30NS055077.; 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 Economía 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.).
• Keywords: Science & Technology; homology modeling; mass spectrometry (MS); Pseudomonas aeruginosa (P. aeruginosa); translation elongation factor; lysine methyltransferase; EF-Tu; Biochemistry & Molecular Biology; S-adenosylmethionine (SAM); Life Sciences & Biomedicine; circular dichroism (CD)
• Research Categories: Health Sciences, General; Biology, Microbiology; Chemistry, Biochemistry

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 notathigher 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 Eft M, 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 thein vivotemperature 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.