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

• Published in: Journal of molecular biology Vol. 288; no. 5; pp. 975 - 987
• Main Authors: Miller, Mitchell D., Cai, Jiwen, Krause, Kurt L.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 21.05.1999
• Subjects: Amino Acid Sequence; Asparagine - metabolism; Binding Sites; Crystallography, X-Ray; divalent cation; DNA-Binding Proteins; Endodeoxyribonucleases - metabolism; endonuclease; Endonucleases - chemistry; magnesium; Magnesium - metabolism; Models, Molecular; Molecular Sequence Data; nuclease; Sequence Homology, Amino Acid; Serratia - chemistry; Water - metabolism; water cluster; endonuclease; divalent cation; magnesium; nuclease; water cluster
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1999.2729

Serratia endonuclease is an important member of a class of magnesium dependent nucleases that are widely distributed in nature. Here, we describe the location and geometry of a magnesium-water cluster within the active site of this enzyme. The sole protein ligand of the magnesium atom is Asn119; this metal ion is also associated with five water molecules to complete an octahedral coordination complex. These water molecules are very well ordered and 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 coordination sphere. Asp86 is not chelated to the magnesium or its surrounding water molecules. Results of kinetics and site-specific mutagenesis experiments suggest that this metal-water cluster contains the catalytic metal ion of this enzyme. All residues which hydrogen bond to the water molecules that coordinate the magnesium atom are conserved in nucleases homologous to Serratia endonuclease, suggesting that the water cluster is a conserved feature of this family of enzymes. We offer a detailed structural comparison to one other nuclease, the homing endonuclease I- PpoI, that has recently been shown, in spite of a lack of sequence homology, to share a similar active site geometry to Serratia endonuclease. Evidence from both of these structures suggests that the magnesium of Serratia nuclease participates in catalysis via an inner sphere mechanism.