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 gmax 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.

A Role for Saccharomyces cerevisiae Tpa1 Protein in Direct Alkylation Repair

“…2D). This is in good agreement with the characteristic absorption peak of chromophores reported by others (33,45,46). However, the Tpa1-mutant, which lacks active site coordination of iron and 2OG, did not produce any spectrum associated with chromophore formation (Fig.…”

“…Although Tpa1 has been characterized before (24 -26), this spectroscopic characteristic was not known in Tpa1. We added excess Fe(II) and 2OG to recombinant Tpa1 and observed an UV-visible band with peak absorption at 530 nm for the native protein, as described previously (33,45,46). This is the first Geneticin 80mg/ml WT tpa1D mag1D tpa1D mag1D stp22D WT tpa1D mag1D tpa1D mag1D stp22D 6-aza-uracil 100mg/ml YPD FIGURE 7.…”

Adaptive Response Enzyme AlkB Preferentially Repairs 1-Methylguanine and 3-Methylthymine Adducts in Double-Stranded DNA

“…One of these enzymes, the adaptive response protein AlkB of E. coli , has been reported to repair alkyl DNA adducts, such as 1-methyladenine (m1A) and 3-methylcytosine (m3C), in both ss- and ds-contexts, although it prefers repairing lesions in single stranded substrates. 4–9 …”

“…4,5 Among the nine AlkB homologs, ABH1 functions as an apyrimidinic/apurinic lyase and nucleic acid demethylase. 6,7 ABH4 8,9 and ABH7 10,11 modify protein substrates and ABH8 12–16 is a tRNA methyltransferase and hydroxylase. ABH5 17–21 and FTO 22–25 have been demonstrated to work on N6-methyladenine (m6A) in RNA or DNA.…”

The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond

“…ALKBH8 (50 -54) is involved in the maturation of tRNA featuring both an S-adenosylmethionine-dependent methyltransferase domain that methylates 5-carboxymethyluridine (cm5U) to 5-methoxycarbonylmethyluridine (mcm5U), and a dioxygenase domain that hydroxylates mcm5U to generate (S)-5-methoxycarbonylhydroxymethyluridine (mchm5U), a common functional modification at the wobble position of tRNA (51,52). The main function of ALKBH4 seems to be actin demethylation (55,56). ALKBH7, which plays a role in alkylationinduced necrosis (57)(58)(59), is believed to act on protein substrates, but their identity is not currently known.…”