Catalytic efficiency of predicted deleterious missense variants of human DNA dioxygenase ABH2

Human DNA dioxygenase ABH2 is a key Fe(II)/α-ketoglutarate-dependent enzyme that removes a broad range of cytotoxic alkylated lesions, including N ¹ -methyladenine (m¹A) and N ³ -methylcytosine (m³C), from double-stranded DNA, thereby supporting genome stability. Naturally occurring missense single nucleotide variants (SNVs) in the ABH2 gene can modulate its catalytic properties and, consequently, the efficiency of direct DNA repair. In this study, we functionally characterized eight ABH2 variants featuring SNVs-associated substitutions G103R, S125L, S188F, F195S, M226T, R248Q, R248W, or T252I, that had been previously (Davletgildeeva et al., 2025) classified as potentially deleterious by multiple prediction algorithms. We quantified kinetic parameters of the corresponding ABH2 variants toward double-stranded DNA substrates containing either m¹A or m³C and compared their catalytic efficiency to that of the wild-type enzyme. For most of the variants, the negative prognosis was justified, as the introduced substitutions, while maintaining the general architecture of the protein globule and having insignificant differences in thermal stability, led to a decrease (G103R, S188F, T252I, and, to a lesser extent, S125L) or even complete loss (R248Q and R248W) of the catalytic activity of the ABH2 enzyme toward both tested damaged nucleobases. At the same time, M226T and F195S variants demonstrated an increase in activity of the enzyme toward m³C, thus contradicting the obtained negative prognosis. Our findings show that naturally occurring ABH2 missense variants can differentially alter the repair efficiency of m¹A and m³C lesions in double-stranded DNA, ranging from partial to complete loss or lesion-selective enhancement of catalytic activity. Structural analysis suggests that these functional effects arise from disruption of local interactions and/or changes in conformational flexibility required to form a catalytically competent enzyme-DNA complex. Overall, these results delineate a diverse functional landscape of naturally occurring ABH2 missense variants and provide a quantitative framework for understanding how sequence variation shapes the enzyme's DNA repair capacity.