Abstract
Paralogs of the bifunctional nuclease, Ribonuclease J (RNase J), demonstrate varied catalytic efficiencies despite extensive sequence and structural similarity. Of the two Staphylococcus aureus RNase J paralogues, RNase J1 is substantially more active than RNase J2. Mutational analysis of active site residues revealed that only H80 and E166 were critical for nuclease activity. Electronic properties of active site residues were further evaluated using density functional theory in conjunction with molecular mechanics. This analysis suggested that multiple residues at the active site can function as Lewis bases or acids in RNase J2. The bond dissociation energy, on the other hand, suggested that the Mn ion in RNase J2, located at a structurally identical location to that in RNase J1, is crucial for overall structural integrity. Structures of mutant enzymes lacking the metal ion were seen to adopt a different orientation between the substrate binding and catalytic domain than wild-type RNase J2. A surprising finding was that the RNase J2 H78 A mutant was five-fold more active than the wild-type enzyme. Structural and biochemical experiments performed in light of this observation revealed that the RNase J2 catalytic mechanism is distinct from both two-metal ion and one-metal ion reaction mechanisms proposed for RNase J nucleases. Different activity levels in RNase J paralogues can thus be ascribed to the diversity in catalytic mechanisms.