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Protein Science (2005), 14:2296-2303. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Specificity inversion of Ochrobactrum anthropi D-aminopeptidase to a D,D-carboxypeptidase with new penicillin binding activity by directed mutagenesis

Michaël Delmarcelle, Marie-Caroline Boursoit, Patrice Filée, Stéphane Lucius Baurin, Jean-Marie Frère and Bernard Joris

Centre d’Ingénierie des Protéines, Institut de Chimie, Universitéde Liège, Sart-Tilman, B-4000 Liège, Belgium

(RECEIVED March 24, 2005; FINAL REVISION May 25, 2005; ACCEPTED May 31, 2005)

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.

Keywords: directed evolution; serine penicillin-recognizing proteins; penicillin binding proteins; Ochrobactrum anthropi D-aminopeptidase; Streptomyces R61 D,D-peptidase

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051475305.

Reprint requests to: Bernard Joris, Centre d’Ingénierie des Protéines, Institut de Chimie, B6a, Universitéde Liège, Sart-Tilman, B- 4000 Liège, Belgium; e-mail: bjoris{at}ulg.ac.be; fax: 00-32-4-366-33-64.


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