Molecular dynamic analysis of the functional role of amino acid residues V99, F124 and S125 of human DNA dioxygenase ABH2

   The ABH2 enzyme belongs to the AlkB-like family of Fe(II)/α-ketoglutarate-dependent dioxygenases. Various non-heme dioxygenases act on a wide range of substrates and have a complex catalytic mechanism involving α-ketoglutarate and an Fe(II) ion as a cofactor. Representatives of the AlkB family catalyze the direct oxidation of alkyl substituents in the nitrogenous bases of DNA and RNA, providing protection against the mutagenic effects of endogenous and exogenous alkylating agents, and also participate in the regulation of the methylation level of some RNAs. DNA dioxygenase ABH2, localized predominantly in the cell nucleus, is specific for double-stranded DNA substrates and, unlike most other human AlkB-like enzymes, has a fairly broad spectrum of substrate specificity, oxidizing alkyl groups of such modified nitrogenous bases as, for example, N 1-methyladenosine, N 3-methylcytidine, 1,N 6-ethenoadenosine and 3,N 4-ethenocytidine.

   To analyze the mechanism underlying the enzyme’s substrate specificity and to clarify the functional role of key active-site amino acid residues, we performed molecular dynamics simulations of complexes of the wild-type ABH2 enzyme and its mutant forms containing amino acid substitutions V99A, F124A and S125A with two types of DNA substrates carrying methylated bases N 1-methyladenine and N 3-methylcytosine, respectively.

   It was found that the V99A substitution leads to an increase in the mobility of protein loops L1 and L2 involved in binding the DNA substrate and changes the distribution of π-π contacts between the side chain of residue F102 and nitrogenous bases located near the damaged nucleotide. The F124A substitution leads to the loss of π-π stacking with the damaged base, which in turn destabilizes the architecture of the active site, disrupts the interaction with the iron ion and prevents optimal catalytic positioning of α-ketoglutarate in the active site. The S125A substitution leads to the loss of direct interaction of the L2 loop with the 5’-phosphate group of the damaged nucleotide, weakening the binding of the enzyme to the DNA substrate. Thus, the obtained data revealed the functional role of three amino acid residues of the active site and contributed to the understanding of the structural-functional relationships in the recognition of a damaged nucleotide and the formation of a catalytic complex by the human ABH2 enzyme.

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