IDENTIFICATION OF CATALYTICALLY RELEVANT AMINO-ACIDS OF THE EXTRACELLULAR SERRATIA-MARCESCENS ENDONUCLEASE BY ALIGNMENT-GUIDED MUTAGENESIS

By sequence alignment of the extracellular Serratia marcescens nucleas e with three related nucleases we have identified seven charged amino acid residues which are conserved in all four sequences. Six of these residues together with four other partially conserved His or Asp resid ues were changed to alanine by site-directed PCR-mediated mutagenesis using a variant of the nuclease gene in which the coding sequence of t he signal peptide was replaced by the coding sequence for an N-termina l affinity tag [Met(His)(6)GlySer]. Four of the mutant proteins showed almost no reduction in nuclease activity but five displayed a 10- to 1000-fold reduction in activity and one (His110Ala) was inactive. Base d upon these results it is suggested that the S.marcescens nuclease em ploys a mechanism in which His110 acts in concert with a Mg2+ ion and three carboxylates (Aspl07, Glu148 and Glu232) as well as one or two b asic amino acid residues (Arg108, Arg152).