Cleavage experiments with deoxythymidine 3′,5′‐bis‐(p‐nitrophenyl phosphate) suggest that the homing endonuclease I‐PpoI follows the same mechanism of phosphodiester bond hydrolysis as the non‐specific Serratia nuclease1

Abstract

We show here that two nucleases, Serratia nuclease and I‐PpoI, with contrasting specificities, i.e. non‐specific vs. highly sequence specific, share a structurally similar active site region with conservation of the catalytically relevant histidine and asparagine residues. On the basis of a comparison of the available structures and biochemical data for wild type and mutant variants of Serratia nuclease and I‐PpoI we propose that both enzymes have a common catalytic mechanism, a proposition that is supported by our finding that both enzymes accept deoxythymidine 3′,5′‐bis‐(p‐nitrophenyl phosphate) as a substrate and cleave it in an identical manner. According to this mechanism a histidine residue functions as a general base and Mg²⁺ bound to an asparagine residue as a Lewis acid in phosphodiester bond cleavage.