Biochemical and kinetic properties of a Type III restriction-modification enzyme Mbo45V from the host-adapted pathogen Mycoplasma bovis

Abstract Type III restriction-modification (RM) enzymes are prominent bacterial defense against bacteriophage and invading foreign DNA that also modulate the host’s epigenetic landscape. Genome analysis of the host-adapted Mycoplasma bovis PG45 that has a very small genome revealed a Type III RM locus comprising one res and three mod genes. We characterized Mbo45V, a representative enzyme encoded by this locus. The enzyme forms a heterotrimeric complex consisting of two Mod subunits and one Res subunit. Mbo45V recognizes the asymmetric sequence 5′-YAATC-3′ (Y = T/C) and cleaves DNA having at least two head-to-head oriented sites ∼26–28 bp away from the recognition site. Methylation of the second adenine of the target site using cofactor S-adenosylmethionine (SAM) protects DNA from restriction, while the SAM analogue sinefungin enhances DNA binding and cleavage. Kinetic studies reveal that Mbo45V exhibits relatively weak DNA binding affinity and an unusually high Km for SAM, indicating low cofactor affinity compared to prototypical enzymes such as EcoP15I. ATPase activity is strongly stimulated by cognate DNA and is inhibited upon methylation of the substrate, suggesting a regulatory interplay between methylation and restriction functions. Comparative analysis indicates that, although Mbo45V shares core mechanistic features with prototypes from Escherichia coli, its kinetic parameters are distinct. These differences likely reflect adaptation to the stable intracellular environment of M. bovis, in contrast to the fluctuating conditions encountered by the enteric bacteria. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵# Joint first author