Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB

Abstract

Nucleic acid damage by environmental and endogenous alkylation reagents creates lesions that are both mutagenic and cytotoxic, with the latter effect accounting for their widespread use in clinical cancer chemotherapy. Escherichia coli AlkB and the homologous human proteins ABH2 and ABH3 (refs 5, 7) promiscuously repair DNA and RNA bases damaged by S(N)2 alkylation reagents, which attach hydrocarbons to endocyclic ring nitrogen atoms (N1 of adenine and guanine and N3 of thymine and cytosine). Although the role of AlkB in DNA repair has long been established based on phenotypic studies, its exact biochemical activity was only elucidated recently after sequence profile analysis revealed it to be a member of the Fe-oxoglutarate-dependent dioxygenase superfamily. These enzymes use an Fe(II) cofactor and 2-oxoglutarate co-substrate to oxidize organic substrates. AlkB hydroxylates an alkylated nucleotide base to produce an unstable product that releases an aldehyde to regenerate the unmodified base. Here we have determined crystal structures of substrate and product complexes of E. coli AlkB at resolutions from 1.8 to 2.3 A. Whereas the Fe-2-oxoglutarate dioxygenase core matches that in other superfamily members, a unique subdomain holds a methylated trinucleotide substrate into the active site through contacts to the polynucleotide backbone. Amide hydrogen exchange studies and crystallographic analyses suggest that this substrate-binding 'lid' is conformationally flexible, which may enable docking of diverse alkylated nucleotide substrates in optimal catalytic geometry. Different crystal structures show open and closed states of a tunnel putatively gating O2 diffusion into the active site. Exposing crystals of the anaerobic Michaelis complex to air yields slow but substantial oxidation of 2-oxoglutarate that is inefficiently coupled to nucleotide oxidation. These observations suggest that protein dynamics modulate redox chemistry and that a hypothesized migration of the reactive oxy-ferryl ligand on the catalytic Fe ion may be impeded when the protein is constrained in the crystal lattice.

Alleviation of 1, N 6 -ethanoadenine genotoxicity by the Escherichia coli adaptive response protein AlkB

“…The fractions containing AlkB⌬N11 were pooled, and purity was established by SDS/PAGE. As previously reported (25), AlkB⌬N11 has similar activity to the full-length protein in standard AlkB assays (data not shown).…”

“…The recently solved x-ray crystal structure (25) shows a particularly malleable region within the nucleotide-recognition lid that may account for the accommodating nature of the protein.…”

Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA

“…The in vivo function of ABH3 is still unclear but it has been shown to repair base lesions in RNA as well as DNA 15 . Although the structures of AlkB (with dT-(1-medA)-dT) 19 and ABH3 (in the absence of DNA or RNA) 20 were solved recently, the molecular mechanism underlying the different substrate preferences of ABH2 and AlkB remains unknown. Furthermore, the structure of ABH2 has, to our knowledge, never been reported.…”

A loop matters for FTO substrate selection

“…So far, several crystal structures of AlkB members, including AlkB, hABH2 and hABH3, have been solved (Sundheim et al, 2006;Yu et al, 2006;Yang et al, 2008). As expected, all the available structures reveal a conserved double-stranded beta-helix (DSBH) fold, termed as jelly-roll motif that coordinates a catalytically active iron center composed of a conserved His-Xaa-Asp/Glu-Xaa-His motif (Hausinger, 2004).…”