Site-Directed Mutagenesis of the Streptomyces R61 Dd-Peptidase. Catalytic Function of the Conserved Residues around the Active Site and a Comparison with Class-a and Class-C Beta-Lactamases
[en] The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65 → Arg mutant with the peptide and thiol ester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20 000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe358 → Leu, Tyr90 → Asn, Thr101 → Asn, Phe164 → Ala, Asp225 → Glu and Asp225 → Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164 → Ala mutant was significantly more unstable than the wild-type enzyme.
Research center :
CIP - Centre d'Ingénierie des Protéines - ULiège
Disciplines :
Microbiology
Author, co-author :
Hadonou, Ayaovi Medard; Université de Liège - ULiège > Centre d'ingénierie des protéines
Wilkin, Jean-Marc; Université de Liège - ULiège > Centre d'ingénierie des protéines
Varetto, Louis ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Joris, Bernard ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Frère, Jean-Marie ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Language :
English
Title :
Site-Directed Mutagenesis of the Streptomyces R61 Dd-Peptidase. Catalytic Function of the Conserved Residues around the Active Site and a Comparison with Class-a and Class-C Beta-Lactamases
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