TY - JOUR

T1 - Role of Individual Amino Acid Residues Directly Involved in Damage Recognition in Active Demethylation by ABH2 Dioxygenase

AU - Davletgildeeva, Anastasiia T.

AU - Tyugashev, Timofey E.

AU - Zhao, Mingxing

AU - Ishchenko, Alexander A.

AU - Saparbaev, Murat

AU - Kuznetsov, Nikita A.

N1 - This work was supported by Russian Ministry of Science and Higher Education project no. 121031300041-4.

PY - 2025

Y1 - 2025

N2 - The enzyme ABH2, one of nine human DNA dioxygenases of the AlkB family, belongs to the superfamily of Fe(II)/α-ketoglutarate-dependent dioxygenases and plays a crucial role in the direct reversal repair of nonbulky alkyl lesions in DNA nucleobases. ABH2 has broad substrate specificity, directly oxidizing DNA damages such as N1-methyladenine, N3-methylcytosine, 1,N6-ethenoadenine, 3,N4-ethenocytosine, and a number of others. In our investigation, we sought to uncover the subtleties of the mechanisms governing substrate specificity in ABH2 by focusing on several critical amino acid residues situated in its active site. To gain insight into the function of this enzyme, we performed a functional mapping of its active site region, concentrating on pivotal residues, participating in forming a damaged binding pocket of the enzyme (Val99 and Ser125), as well as the residues directly involved in interactions with damaged bases, namely Arg110, Phe124, Arg172, and Glu175. To support our experimental data, we conducted a series of molecular dynamics simulations, exploring the interactions between the ABH2 mutant forms, bearing corresponding substitutions and DNA substrates, and harboring various types of methylated bases, specifically N1-methyladenine or N3-methylcytosine. The comparative studies revealed compelling data indicating that alterations in most of the studied amino acid residues significantly influence both the binding affinity of the enzyme for DNA and its catalytic efficiency. Intriguingly, the findings suggest that the mutations impact the catalytic activity of ABH2 to a greater extent than its ability to associate with DNA strands. Collectively, these results show how changes to the active site affect molecular dynamics and reaction kinetics, improving our understanding of the substrate recognition process in this pivotal enzyme.

AB - The enzyme ABH2, one of nine human DNA dioxygenases of the AlkB family, belongs to the superfamily of Fe(II)/α-ketoglutarate-dependent dioxygenases and plays a crucial role in the direct reversal repair of nonbulky alkyl lesions in DNA nucleobases. ABH2 has broad substrate specificity, directly oxidizing DNA damages such as N1-methyladenine, N3-methylcytosine, 1,N6-ethenoadenine, 3,N4-ethenocytosine, and a number of others. In our investigation, we sought to uncover the subtleties of the mechanisms governing substrate specificity in ABH2 by focusing on several critical amino acid residues situated in its active site. To gain insight into the function of this enzyme, we performed a functional mapping of its active site region, concentrating on pivotal residues, participating in forming a damaged binding pocket of the enzyme (Val99 and Ser125), as well as the residues directly involved in interactions with damaged bases, namely Arg110, Phe124, Arg172, and Glu175. To support our experimental data, we conducted a series of molecular dynamics simulations, exploring the interactions between the ABH2 mutant forms, bearing corresponding substitutions and DNA substrates, and harboring various types of methylated bases, specifically N1-methyladenine or N3-methylcytosine. The comparative studies revealed compelling data indicating that alterations in most of the studied amino acid residues significantly influence both the binding affinity of the enzyme for DNA and its catalytic efficiency. Intriguingly, the findings suggest that the mutations impact the catalytic activity of ABH2 to a greater extent than its ability to associate with DNA strands. Collectively, these results show how changes to the active site affect molecular dynamics and reaction kinetics, improving our understanding of the substrate recognition process in this pivotal enzyme.

KW - DNA dioxygenase ABH2

KW - DNA methylation

KW - DNA repair

KW - MD modeling

KW - conformational dynamics

KW - functional role of amino acid residues

UR - https://www.scopus.com/pages/publications/105011717428

UR - https://www.mendeley.com/catalogue/97493a28-0ba9-3b10-948e-94cb0bcb67da/

U2 - 10.3390/ijms26146912

DO - 10.3390/ijms26146912

M3 - Article

C2 - 40725158

VL - 26

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 14

M1 - 6912

ER -