Specificity inversion of Ochrobactrum anthropi D-aminopeptidase to a D,D-carboxypeptidase with new penicillin binding activity by directed mutagenesis.
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.
Delmarcelle
Michaël
M
Centre d'Ingénierie des Protéines, Institut de Chimie, B6a, Universitéde Liège, Sart-Tilman, B- 4000 Liège, Belgium.
Boursoit
Marie-Caroline
MC
Filée
Patrice
P
Baurin
Stéphane Lucius
SL
Frère
Jean-Marie
JM
Joris
Bernard
B
D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D016297:Mutagenesis, Site-Directed; D020621:Ochrobactrum anthropi / Q000201:enzymology*; D046915:Penicillin-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D046915:Penicillin-Binding Proteins; D000626:Aminopeptidases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase