AlkB Dioxygenase Preferentially Repairs Protonated Substrates SPECIFICITY AGAINST EXOCYCLIC ADDUCTS AND MOLECULAR MECHANISM OF ACTION

作者：Maciejewska Agnieszka M^*; Poznanski Jaroslaw; Kaczmarska Zuzanna; Krowisz Beata; Nieminuszczy Jadwiga; Polkowska Nowakowska Agnieszka; Grzesiuk Elzbieta; Kusmierek Jaroslaw T

来源：Journal of Biological Chemistry, 2013, 288(1): 432-441.

DOI：10.1074/jbc.M112.353342

Efficient repair by Escherichia coli AlkB dioxygenase of exo-cyclic DNA adducts 3, N-4-ethenocytosine, 1, N-6-ethenoadenine, 3, N-4-alpha-hydroxyethanocytosine, and reported here for the first time 3, N-4-alpha-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-alpha-hydroxyethanocytosine or 3, N-4-alpha-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.

• 出版日期2013-1-4