PDBsum entry 2fdh

Oxidoreductase/DNA

PDB id: 2fdh

Contents

Protein chain

200 a.a.

DNA/RNA

Ligands

AKG

Metals

_MN

Waters ×225

References listed in PDB file

Key reference

Title

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

Authors

B.Yu, W.C.Edstrom, J.Benach, Y.Hamuro, P.C.Weber, B.R.Gibney, J.F.Hunt.

Ref.

Nature, 2006, 439, 879-884. [DOI no: 10.1038/nature04561]

PubMed id

16482161

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.

Figure 1.
Figure 1: Crystal structure of the anaerobic Michaelis complex of E. coli AlkB- Delta-N11 with Fe(ii), 2OG and a methylated trinucleotide. a, Stereo ribbon diagram with the backbone coloured and the 2° structural elements labelled according to subdomain organization (with the N-terminal extension (N) in yellow, nucleotide-recognition lid (L) in blue, and catalytic core (C) in green as in the sequence-structure alignment in Supplementary Fig. S2A). Most of the blue segment is protected against amide H/D exchange by dT-(1-me-dA)-dT substrate binding (Supplementary Fig. S2A). The sphere representing the Fe cofactor is coloured orange, whereas atoms in 2OG and dT-(1-me-dA)-dT are coloured according to atomic identity (carbon, white; oxygen, red; nitrogen, blue; and phosphorous, orange). Invariant side chains in Fe-2OG dioxygenases are coloured red or magenta depending on whether they interact with Fe or 2OG, respectively. b, Least-squares superposition of 80 out of 211 C atoms in AlkB with a root mean square deviation of 2.1 Å with the equivalent atoms in the taurine oxidase TauD^16 (Protein Data Bank 1OS7; protein backbone and ligands coloured red). In TauD, the 1-carboxylate of 2OG interacts with Fe in the alternative geometry observed in crystal structures of some Fe-2OG dioxygenases before O[2]-analogue binding18.

Figure 3.
Figure 3: Stereo pairs showing active site stereochemistry in alternative ligand complexes of AlkB- Delta-N11. Ligands and side chains are coloured according to atomic identity as in Fig. 1. a, The anaerobic Michaelis complex has all of the octahedral coordination sites on the Fe cofactor occupied by protein or 2OG atoms except for a single site occupied by a crystallographic water, which must be replaced by O[2] to initiate oxidation (Supplementary Fig. S1). b, Equivalent view of the structure co-crystallized with Fe, succinate and dT-(1-me-dA)-dT (Supplementary Table S2). c, Equivalent view of the structure in which the anaerobic Michaelis complex was exposed to oxygen for 2 h. Unbiased electron density maps (Supplementary Fig. S5B) and occupancy refinement (Supplementary Table S3) both support the conclusion that most of the 2OG has been oxidized to succinate but that the adenine base remains largely methylated after short-term in situ oxidation. The alternative ligands are shown in semi-transparent rendering with the degree of transparency scaled according to refined occupancy. d, Refined 2F[o] - F[c] (green) and F[o] - F[c] (red) electron density maps for the structure shown in c contoured at +1 and -3 , respectively. There are no F[o] - F[c] peaks + 3 in this region.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2006, 439, 879-884) copyright 2006.