title: Spectroscopic and magnetic studies of wild-type and mutant forms of the Fe(II)- and 2-oxoglutarate-dependent decarboxylase ALKBH4.
abstract: The Fe(II)/2OG (2-oxoglutarate)-dependent dioxygenase superfamily comprises proteins that couple substrate oxidation to decarboxylation of 2OG to succinate. A member of this class of mononuclear non-haem Fe proteins is the Escherichia coli DNA/RNA repair enzyme AlkB. In the present work, we describe the magnetic and optical properties of the yet uncharacterized human ALKBH4 (AlkB homologue). Through EPR and UV-visible spectroscopy studies, we address the Fe-binding environment of the proposed catalytic centre of wild-type ALKBH4 and an Fe(II)-binding mutant. We could observe a novel unusual Fe(III) high-spin EPR-active species in the presence of sulfide with a g(max) of 8.2. The Fe(II) site was probed with NO. An intact histidine-carboxylate site is necessary for productive Fe binding. We also report the presence of a unique cysteine-rich motif conserved in the N-terminus of ALKBH4 orthologues, and investigate its possible Fe-binding ability. Furthermore, we show that recombinant ALKBH4 mediates decarboxylation of 2OG in absence of primary substrate. This activity is dependent on Fe as well as on residues predicted to be involved in Fe(II) co-ordination. The present results demonstrate that ALKBH4 represents an active Fe(II)/2OG-dependent decarboxylase and suggest that the cysteine cluster is involved in processes other than Fe co-ordination.
authors.affiliation: Department of Molecular Biosciences, University of Oslo, Blindern, NO-0316 Oslo, Norway.
authors.forename: Linn G
authors.initials: LG
authors.lastname: Bjørnstad
authors.forename: Giorgio
authors.initials: G
authors.lastname: Zoppellaro
authors.forename: Ane B
authors.initials: AB
authors.lastname: Tomter
authors.forename: Pål Ø
authors.initials: PØ
authors.lastname: Falnes
authors.forename: K Kristoffer
authors.initials: KK
authors.lastname: Andersson
mesh_terms: D000071501:AlkB Homolog 4, Lysine Demethylase; D020816:Amino Acid Motifs; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism*; D009154:Mutation; D013056:Spectrophotometry, Ultraviolet
chemical_list: D007656:Ketoglutaric Acids; D007501:Iron; D049308:Dioxygenases; C557720:ALKBH4 protein, human; D000071501:AlkB Homolog 4, Lysine Demethylase; D002262:Carboxy-Lyases