Reactivation of Serratia marcescens Endonuclease NucSma(H89G) by Hydroxilamine

Abstract

Serratia marcescens endonuclease is the most nonspecific nuclease known. This nuclease refers to enzymes that can cleave both DNA and RNA. The active site of this enzyme is characterized by the H–N–H motif, but more important functions have been shown for the first amino acid of this motif in Serratia marcescens nuclease. It is known that histidine is essential in catalytic activity of many nucleases. Histidine functions as a general base that activates a water molecule for a nucleophilic attack at the diester linkages phosphorus atom and causes its rupture. It has been demonstrated that glycine-histidine substitution in Serratia marcescens endonuclease leads to inactivation of the enzyme. At present, the method of chemical recovery of enzymes has become very widespread. Upon addition of certain low molecular compounds, which are similar in chemical properties to the lost amino acid residues, to the medium with the inactive mutant enzyme, the activity of this enzyme has been restored. The recovery effect has been observed due to the fact that the original amino acids are replaced by a low molecular weight as a result of mutations. In this work we have been able to restore the activity of Serratia marcescens mutant endonuclease by adding hydroxylamine, where histidine at position 89 is replaced by glycine.

Keywords

• endonuclease
• plasmid
• hydroxylamine
• histidine
• imidazole