Identification of ALKBH6 as a nucleotide demethylase with a distinct substrate preference

AlkB homolog (ALKBH) family enzymes remove the methyl groups from a variety of substrates including ssDNA and dsDNA, RNA, and proteins. There are eight different ALKBH genes (ALKBH1-8) encoded in the human genome. However, the identity of the substrate of ALKBH6 has remained elusive. In this study, through biochemical experiments and mass spectrometry analysis, we demonstrate that ALKBH6 effectively demethylates N-7-methyl-GMP, and N-1-methyl-adenosine monophosphate. We observed that the presence of a phosphate group in the substrate molecule is essential for ALKBH6 activity as it showed no activity toward methylated bases or the methylated nucleosides. Enzyme kinetic analysis led to the identification of the catalytic and substrate binding residues of ALKBH6 and revealed that succinate and oncometabolite 2-hydroxyglutarate can act as inhibitors of this enzyme. By using mass spectrometry analysis, immunofluorescence microscopy, and ELISA, we confirmed that N-7-methyl-GMP and N-1-methyl-AMP are also the endogenous substrates of ALKBH6. This functional characterization of ALKBH6 broadens the substrate range of ALKBH family proteins and positions ALKBH6 as one of the key players involved in sanitizing the N-methylated nucleotides.