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

• Published in: Protein science 2005-09, Vol.14 (9), p.2296-2303
• Main Authors: Delmarcelle, Michaël, Boursoit, Marie-Caroline, Filée, Patrice, Baurin, Stéphane Lucius, Frère, Jean-Marie, Joris, Bernard
• Format: Article
• Language: English
• Subjects: Aminopeptidases - chemistry; Aminopeptidases - genetics; Aminopeptidases - metabolism; Binding Sites; Biochemistry; Biochemistry, biophysics & molecular biology; Biochimie, biophysique & biologie moléculaire; biophysics; directed evolution; Life sciences; molecular biology; Mutagenesis, Site-Directed; Ochrobactrum anthropi; Ochrobactrum anthropi - enzymology; Ochrobactrum anthropi D‐aminopeptidase; penicillin binding proteins; Penicillin-Binding Proteins - genetics; Penicillin-Binding Proteins - metabolism; Protein Conformation; Protein Engineering - methods; Sciences du vivant; serine penicillin‐recognizing proteins; 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; Streptomyces - enzymology; Streptomyces R61 D,D‐peptidase; Structure-Activity Relationship; Substrate Specificity
• ISSN: 0961-8368; 1469-896X; 1469-896X
• 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 β‐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.