Individual Contributions of Amido Acid Residues Tyr122, Ile168, and Asp173 to the Activity and Substrate Specificity of Human DNA Dioxygenase ABH2

Cells. 2023 Jul 13;12(14):1839. doi: 10.3390/cells12141839.

Abstract

Human Fe(II)/α-ketoglutarate-dependent dioxygenase ABH2 plays a crucial role in the direct reversal repair of nonbulky alkyl lesions in DNA nucleobases, e.g., N1-methyladenine (m1A), N3-methylcytosine (m3C), and some etheno derivatives. Moreover, ABH2 is capable of a less efficient oxidation of an epigenetic DNA mark called 5-methylcytosine (m5C), which typically is a specific target of DNA dioxygenases from the TET family. In this study, to elucidate the mechanism of the substrate specificity of ABH2, we investigated the role of several active-site amino acid residues. Functional mapping of the lesion-binding pocket was performed through the analysis of the functions of Tyr122, Ile168, and Asp173 in the damaged base recognition mechanism. Interactions of wild-type ABH2, or its mutants Y122A, I168A, or D173A, with damaged DNA containing the methylated base m1A or m3C or the epigenetic marker m5C were analyzed by molecular dynamics simulations and kinetic assays. Comparative analysis of the enzymes revealed an effect of the substitutions on DNA binding and on catalytic activity. Obtained data clearly demonstrate the effect of the tested amino acid residues on the catalytic activity of the enzymes rather than the DNA-binding ability. Taken together, these data shed light on the molecular and kinetic consequences of the substitution of active-site residues for the mechanism of the substrate recognition.

Keywords: DNA dioxygenase ABH2; DNA methylation; DNA repair; conformational dynamics; fluorescent spectroscopy; presteady-state kinetics; stopped-flow.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids
  • DNA / metabolism
  • DNA Repair Enzymes / metabolism
  • Dioxygenases* / metabolism
  • Humans
  • Substrate Specificity

Substances

  • Dioxygenases
  • DNA Repair Enzymes
  • DNA
  • Amino Acids

Grants and funding

This work was partially supported by the Russian Federal Ministry of Science and Higher Education (project no. 121031300041-4 for N.A.K.), by Electricité de France (grant nos. RB 2020-02 and RB 2021-05 to M.S.), by the French National Research Agency (grant no. ANR-18-CE44-0008 to A.A.I.), and Fondation ARC (grant no. PJA-2021060003796 to A.A.I.). The part of the work involving the analysis of the ABH2 conformational dynamics and activity was specifically funded by the Russian Science Foundation grant no. 21-14-00018.