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Nucleic Acids Research, 1994, Vol. 22, No. 16 3280-3287
© 1994

ENZYMOLOGY

Identification of catalytically relevant amino acids of the extracellular Serratia marcescens endonuclease by alignment-guided mutagenesis

Peter Friedhoff, Oleg Gimadutdinow1 and Alfred Pingoud*

Institut für Biochemie, Justus-Liebig-Universität Heinrich-Buff-Ring 58, D-35393 Giessen, Germany 1University of Kazan St Lenin 18, 420008 Kazan 8, Russia

*To whom correspondence should be addressed

Received August 5, 1994. Revised August 15, 1994. Accepted August 15, 1994.

By sequence alignment of the extracellular Serratia marcescens nuclease 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 residues were changed to alanine by sitedirected PCR-mediated mutagenesis using a variant of the nuclease gene in which the coding sequence of the signal peptide was replaced by the coding sequence for an N-terminal affinity tag [Met(His)6GlySer]. 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. Based upon these results it is suggested that the S.marcescens nuclease employs a mechanism in which His110 acts in concert with a Mg2+ ion and three carboxylates (Asp107, Glu148 and Glu232) as well as one or two basic amino acid residues (Arg108, Arg152).


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