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. 2013 Jan 4;288(1):432-41.
doi: 10.1074/jbc.M112.353342. Epub 2012 Nov 12.

AlkB dioxygenase preferentially repairs protonated substrates: specificity against exocyclic adducts and molecular mechanism of action

Agnieszka M Maciejewska  1 , Jaroslaw Poznanski, Zuzanna Kaczmarska, Beata Krowisz, Jadwiga Nieminuszczy, Agnieszka Polkowska-Nowakowska, Elzbieta Grzesiuk, Jaroslaw T Kusmierek
Affiliations

AlkB dioxygenase preferentially repairs protonated substrates: specificity against exocyclic adducts and molecular mechanism of action

Agnieszka M Maciejewska et al. J Biol Chem. .

Abstract

Efficient repair by Escherichia coli AlkB dioxygenase of exocyclic DNA adducts 3,N(4)-ethenocytosine, 1,N(6)-ethenoadenine, 3,N(4)-α-hydroxyethanocytosine, and reported here for the first time 3,N(4)-α-hydroxypropanocytosine requires higher Fe(II) concentration than the reference 3-methylcytosine. The pH optimum for the repair follows the order of pK(a) values for protonation of the adduct, suggesting that positively charged substrates favorably interact with the negatively charged carboxylic group of Asp-135 side chain in the enzyme active center. This interaction is supported by molecular modeling, indicating that 1,N(6)-ethenoadenine and 3,N(4)-ethenocytosine are bound to AlkB more favorably in their protonated cationic forms. An analysis of the pattern of intermolecular interactions that stabilize the location of the ligand points to a role of Asp-135 in recognition of the adduct in its protonated form. Moreover, ab initio calculations also underline the role of substrate protonation in lowering the free energy barrier of the transition state of epoxidation of the etheno adducts studied. The observed time courses of repair of mixtures of stereoisomers of 3,N(4)-α-hydroxyethanocytosine or 3,N(4)-α-hydroxypropanocytosine are unequivocally two-exponential curves, indicating that the respective isomers are repaired by AlkB with different efficiencies. Molecular modeling of these adducts bound by AlkB allowed evaluation of the participation of their possible conformational states in the enzymatic reaction.

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Figures

FIGURE 1.
FIGURE 1.
Structures of studied adducts to DNA bases. Shown are methylated (m3C) and lipid peroxidation products, exocyclic unsaturated (ϵC and ϵA) and exocyclic saturated (HEC and HPC). Note that Cα of HEC and HPC is chiral.
FIGURE 2.
FIGURE 2.
HPLC profiles of HPC repair by AlkB protein. A, substrate TT(HPC)TT pentamer (retention time, 13.7 min) containing 12% unmodified TTCTT (retention time,11.9 min). B, 4-min reaction, 98% repair. AlkB reaction conditions were as follows: 50 mm HEPES buffer, pH of 7.5, 1 mm dithiothreitol, 100 μm Fe(NH4)2(SO4)2, 50 μm αKG, 500 pmol of TT(HPC)TT substrate, and 50 pmol of AlkB protein. The HPLC peak occurring at 9.6 min represents dithiothreitol. mAU, arbitrary units.
FIGURE 3.
FIGURE 3.
Interdependence between optimal concentrations of H+ and Fe(II) cations for repair of various adducts by AlkB protein.
FIGURE 4.
FIGURE 4.
Pseudo first order kinetics of AlkB repair. The kinetics of the repair processes for ϵA (A) and HPC (B) are shown. The linearity observed for ϵA repair identifies, in accordance with Equation 2, a pseudo first order reaction. In the case of HPC, two evident asymptotes clearly identify two species visibly differing in their repair rates. Errors bars represent S.D.
FIGURE 5.
FIGURE 5.
Kinetics of HPC stereoisomer mixture repair by various amounts of AlkB. Marks of a given type represent sets of experimental data, and solid lines follow the single substrate model (A) and two-substrate model (B) fitted to the same data. The visibly better agreement was obtained for the two-substrate model (see Table 1 for details), pointing to the substantial difference in repair rates of both stereoisomers (see Fig. 8 for molecular basis of that effect). Error bars represent S.D. estimated for those experiments that were repeated three times. The figure shows the initial 30 min of reactions. See supplemental Fig. S2 for the full course of the reactions.
FIGURE 6.
FIGURE 6.
Proposed structures of AlkB complexed with ϵA and ϵC. The coordinates for neutral and charged substrates were obtained after 20 rounds of the simulated annealing procedure started from the accessible structure of m1A bound to AlkB (Protein Data Bank code 3I2O) (27). Asp-135, postulated to be crucial for recognition of substrates in their cationic forms, is shown in stick representation, and Fe(II) is marked by a pink sphere of the appropriate radius.
FIGURE 7.
FIGURE 7.
ϵA location in AlkB active center. A and C, ribbon models. B and D, protein surface-colored according to electrostatic potential distribution from red (−250 kJ/mol) to blue (+250 kJ/mol).
FIGURE 8.
FIGURE 8.
Stereoview of proposed structures of AlkB complexed with S (A and C) and R (B and D) stereoisomers of charged HEC (A and B) and HPC (C and D) substrates in anti conformation. Results were obtained from 15-ns molecular dynamics simulations. See supplemental Fig. S8 for proposed structures of complexes with those ligands in syn conformation.
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