Functional and structural evolution of 5-methylpyrimidine dioxygenases (5mYOXs)

5mYOXs – also known as the TET/JBP-like enzymes – are iron (II)/2-oxoglutarate-dependent dioxygenases that catalyze oxidation of 5-methylpyrimidine on nucleic acid polymers to 5-hydroxylpyrimidine (5hmY), and in some cases 5-formylpyrimidine and 5-carboxylpyrimidine (Parker, M. J. et al. (2020), Comprehensive Natural Products III, Elsevier, 5.19, 465-488). In eukaryotes, the modified bases function as epigenetic markers and/or as intermediates in DNA demethylation. In bacterial viruses, generation of 5hmY is an initial step in hypermodification of DNA for evasion of host cell immune responses. Despite the known biological importance of 5mYOX superfamily enzymes, very few members have been characterized to-date. We aim to explore the functional and structural evolution of the 5mYOX superfamily by biochemically characterizing divergent enzymes from all domains of life. We are addressing fundamental questions related to substrate specificity differences observed with different 5mYOXs (e.g., 5-methylcytosine vs. thymine as precursor bases, single- or double-stranded DNA or RNA). We are also exploring the regulatory mechanisms that lead certain members of this family to perform iterative oxidation, while others halt oxidation at 5hmY. Results from these studies are guiding us toward a more comprehensive understanding of the structural domains responsible for discrete enzymatic functions and the evolutionary trajectory of the 5mYOX superfamily. We are actively expanding these aims to probe the broader biological roles of 5mYOX enzymes in the life cycles of diverse organisms. We are also interested in employing these enzymes in next-generation sequencing applications for mapping 5mC and hypermodified derivatives at single-base resolution across entire genomes.