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 nuclease.
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.
Friedhoff
P
P
Institut für Biochemie (FB 15), Justus-Liebig-Universität, Giessen, Germany.
Franke
I
I
Krause
K L
KL
Pingoud
A
A
D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D004952:Esters / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007438:Introns; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013936:Thymidine / Q000031:analogs & derivatives* / Q000378:metabolism