Specificity inversion of Ochrobactrum anthropi D-aminopeptidase to a D,D-carboxypeptidase with new penicillin binding activity by directed mutagenesis

Protein Sci. 2005 Sep;14(9):2296-303. doi: 10.1110/ps.051475305.


The serine penicillin-recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to beta-lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D-aminopeptidase and of the Streptomyces R61 D,D-carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D-aminopeptidase lost its original activity in favor of a new penicillin-binding activity. D-aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild-type enzyme. By a second step site-directed mutagenesis, the specificity of the Ochrobactrum anthropi D-aminopeptidase was inverted to a D,D-carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin-recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin-recognizing proteins are discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminopeptidases / chemistry
  • Aminopeptidases / genetics
  • Aminopeptidases / metabolism*
  • Binding Sites
  • Mutagenesis, Site-Directed
  • Ochrobactrum anthropi / enzymology*
  • Penicillin-Binding Proteins / genetics
  • Penicillin-Binding Proteins / metabolism
  • Protein Conformation
  • Protein Engineering / methods
  • Serine-Type D-Ala-D-Ala Carboxypeptidase / chemistry
  • Serine-Type D-Ala-D-Ala Carboxypeptidase / genetics
  • Serine-Type D-Ala-D-Ala Carboxypeptidase / metabolism*
  • Streptomyces / enzymology
  • Structure-Activity Relationship
  • Substrate Specificity


  • Penicillin-Binding Proteins
  • Aminopeptidases
  • Serine-Type D-Ala-D-Ala Carboxypeptidase