title: Evidence for conformational changes in the yeast deoxyhypusine hydroxylase Lia1 upon iron displacement from its active site.
abstract: The unique amino acid hypusine is formed exclusively in eIF5A by the successive action of deoxyhypusine synthase and deoxyhypusine hydroxylase (yeast Lia1, human DOHH). Although the first enzyme has been extensively studied, both Lia1 structure and the mechanism of action remain unclear. Hence, a multi-approach was used to evaluate Lia1 catalysis, metal/substrate binding, structural conformation and stability. Mutational analyses of Lia1 revealed fine differences in the mode of substrate binding between the human and yeast counterparts. Like human DOHH, recombinant Lia1 is an iron metalloenzyme. Iron is essential for enzyme activity since its loss renders the enzyme totally inactive. The separation of iron-free and iron-bound forms by gel filtration and native electrophoresis suggests differences in Lia1 tertiary structure related to the iron binding. The ability of Lia1 to undergo conformational changes prompted us to use a set of complementary spectroscopic approaches and SAXS to obtain detailed information on the processes underlying dissociation of iron from Lia1 at different levels of the protein organization. The additive effect of weak interactions, especially within the metal center, resulted in an active enzyme in a stabilized and compact three-dimensional fold. Loss of tertiary contacts upon iron displacement led to an elongated conformation of Lia1, in which the N- and C-terminal domains are no longer in close proximity to guarantee the proper orientation of the active groups within the active site pocket. Our results demonstrate an essential structural role for iron binding in addition to its contribution to the catalysis of hypusine formation in the eIF-5A precursor.
authors: Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University-UNESP, Rodovia Araraquara-Jaú Km01, Araraquara, SP, 14801-902, Brazil.
authors: Veridiana S P
authors: VS
authors: Cano
authors: Francisco Javier
authors: FJ
authors: Medrano
authors: Myung Hee
authors: MH
authors: Park
authors: Sandro R
authors: SR
authors: Valentini
mesh_terms: D020134:Catalytic Domain; D004795:Enzyme Stability; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D007501:Iron; D006899:Mixed Function Oxygenases; C047991:deoxyhypusine hydroxylase