title: Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea.
abstract: Translation initiation factor 5A (IF5A) is essential and highly conserved in Eukarya (eIF5A) and Archaea (aIF5A). The activity of IF5A requires hypusine, a posttranslational modification synthesized in Eukarya from the polyamine precursor spermidine. Intracellular polyamine analyses revealed that agmatine and cadaverine were the main polyamines produced inHaloferax volcaniiin minimal medium, raising the question of how hypusine is synthesized in this halophilic Archaea. Metabolic reconstruction led to a tentative picture of polyamine metabolism and aIF5A modification inHfx. volcaniithat was experimentally tested. Analysis of aIF5A fromHfx. volcaniiby LC-MS/MS revealed it was exclusively deoxyhypusinylated. Genetic studies confirmed the role of the predicted arginine decarboxylase gene(HVO_1958)in agmatine synthesis. The agmatinase-like gene(HVO_2299)was found to be essential, consistent with a role in aIF5A modification predicted by physical clustering evidence. Recombinant deoxyhypusine synthase (DHS) fromS. cerevisiaewas shown to transfer 4-aminobutyl moiety from spermidine to aIF5A fromHfx. volcanii in vitro.However, at least under conditions tested, this transfer was not observed with theHfx. volcaniiDHS. Furthermore, the growth ofHfx. volcaniiwas not inhibited by the classical DHS inhibitor GC7. We propose a model of deoxyhypusine synthesis inHfx. volcaniithat differs from the canonical eukaryotic pathway, paving the way for further studies.
authors.affiliation: Department of Microbiology and Cell Science, Institute for Food and Agricultural Sciences and Genetic Institute, University of Florida, P.O. Box 110700, Gainesville, FL 32611-0700, USA.
authors.forename: Laurence
authors.identifier: 0000-0002-4588-1070
authors.initials: L
authors.lastname: Prunetti
authors.affiliation: Gene Center and Department for Biochemistry, University of Munich, 81377 Munich, Germany; Center for Integrated Protein Science Munich, University of Munich, 81377 Munich, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Graf
authors.affiliation: Department of Microbiology and Cell Science, Institute for Food and Agricultural Sciences and Genetic Institute, University of Florida, P.O. Box 110700, Gainesville, FL 32611-0700, USA.
authors.forename: Ian K
authors.initials: IK
authors.lastname: Blaby
authors.affiliation: Faculty of Science and Technology, Institute of Technology, University of Tartu, Tartu, Estonia.
authors.forename: Lauri
authors.identifier: 0000-0001-6906-7113
authors.initials: L
authors.lastname: Peil
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, 91 N. Eagleville Rd, Storrs, CT 06269, USA.
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Makkay
authors.affiliation: Gene Center and Department for Biochemistry, University of Munich, 81377 Munich, Germany; Center for Integrated Protein Science Munich, University of Munich, 81377 Munich, Germany.
authors.forename: Agata L
authors.initials: AL
authors.lastname: Starosta
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, 91 N. Eagleville Rd, Storrs, CT 06269, USA.
authors.forename: R Thane
authors.initials: RT
authors.lastname: Papke
authors.affiliation: Institute of Environmental Microbiology, Kyowa Kako Co. Ltd., Tadao 2-15-5, Machida 194-0035, Japan.
authors.forename: Tairo
authors.initials: T
authors.lastname: Oshima
authors.affiliation: Gene Center and Department for Biochemistry, University of Munich, 81377 Munich, Germany; Center for Integrated Protein Science Munich, University of Munich, 81377 Munich, Germany.
authors.forename: Daniel N
authors.initials: DN
authors.lastname: Wilson
authors.affiliation: Department of Microbiology and Cell Science, Institute for Food and Agricultural Sciences and Genetic Institute, University of Florida, P.O. Box 110700, Gainesville, FL 32611-0700, USA.
authors.forename: Valérie
authors.identifier: 0000-0002-9955-3785
authors.initials: V
authors.lastname: de Crécy-Lagard
mesh_terms: D019843:Archaeal Proteins / Q000378:metabolism*; D002853:Chromatography, Liquid; D019622:Haloferax volcanii / Q000201:enzymology* / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry
chemical_list: D019843:Archaeal Proteins; D010448:Peptide Initiation Factors; C100028:hypusine; D008239:Lysine