Uncovering ParB-dependent and -independent subclasses of T-dioxygenases from bacteriophage

5-Methylpyrimidine dioxygenases (5mYOXs) are iron (II)/2-oxoglutarate-dependent enzymes that catalyze the postreplicative oxidation of DNA 5-methylpyrimidines. Here, we define two subclasses of phage thymine (T) 5mYOXs: a stand-alone enzyme and a second requiring an activator. Using bioinformatic tools, we show that the activator is homologous to the bacterial chromosomal segregation (CS) protein ParB, retaining the N - terminal nucleotide-binding domain (NBD), responsible for CTP binding and hydrolysis in CS, and the C-terminal dimerization domain (CTD), but lacking an obvious DNA-binding domain. In vivo, we demonstrate that ParB activates its cognate 5mYOX with relative specificity and that both NBD and CTD are required for function. Unlike CS-ParBs, mutation of conserved NTP-binding/hydrolysis residues does not affect the role of 5mYOX-associated ParB, suggesting a lack of CTP requirement or a regulatory mechanism not captured under our conditions. For 5mYOXs, we define subclass-specific domains essential for T oxidation and provide evidence for abolishing ParB dependency upon swapping a variable insert from a ParB-independent 5mYOX into a dependent one. In vitro, reconstitution of subclass representatives, 5mYOX97 and 176, confirms their activity as postreplicative T-dioxygenases. Both enzymes function on a wide range of DNA substrates single and double-stranded (ds), linear, and circular. The enzymes differ in their sequence preference and support iterative oxidation of T and 5-methylcytosine. Notably, 5mYOX176 requires activation by ParB176 only when acting on dsDNA. These findings establish an activator-dependent subclass within the iron (II)/2-oxoglutarate-dependent dioxygenase superfamily and expand the functional landscape of both 5mYOX and ParBs, suggesting regulatory mechanisms for T oxidation in bacteriophage.