The active site of Serratia endonuclease contains a conserved magnesium-water cluster

Serratia endonuclease is an important member of a class of magnesium depend ent nucleases that are widely distributed in nature. Here, we describe the location and geometry of a magnesium-water cluster within the active site o f this enzyme. The sole protein ligand of the magnesium atom is Asn119; thi s metal ion is also associated with five water molecules to complete an oct ahedral coordination complex. These water molecules are very well ordered a nd there is no evidence of rotational disorder or motion. Glu127 and His89 are located nearby and each is hydrogen bonded to water molecules in the co ordination sphere. Asp86 is not chelated to the magnesium or its surroundin g water molecules. Results of kinetics and site-specific mutagenesis experi ments suggest that this metal-water cluster contains the catalytic metal io n of this enzyme. All residues which hydrogen bond to the water molecules t hat coordinate the magnesium atom are conserved in nucleases homologous to Serratia endonuclease, suggesting that the water cluster is a conserved fea ture of this family of enzymes. We offer a detailed structural comparison t o one other nuclease, the homing endonuclease I-PpoI, that has recently bee n shown, in spite of a lack of sequence homology, to share a similar active site geometry to Serratia endonuclease. Evidence from both of these struct ures suggests that the magnesium of Serratia nuclease participates in catal ysis via an inner sphere mechanism. (C) 1999 Academic Press.