[en] Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp271 was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp271 residue, though almost invariant among the beta-lactamases of classes A and C and the low-Mr penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp233 into Leu and Ser strongly decreased the enzymic activity, the affinity for beta-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant DD-peptidase thus behaved as a weak beta-lactamase.
Research Center/Unit :
CIP - Centre d'Ingénierie des Protéines - ULiège
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Bourguignon-Bellefroid, Catherine; Université de Liège - ULiège > Institut de Chimie > Laboratoire d'Enzymologie / Centre d'Ingénierie des Proteines
Wilkin, Jean-Marc; Université de Liège - ULiège > Institut de Chimie > Laboratoire d'Enzymologie / Centre d'Ingénierie des Proteines
Joris, Bernard ; Université de Liège - ULiège > Institut de Chimie > Laboratoire d'Enzymologie / Centre d'Ingénierie des Proteines
Aplin, Robin T.; University of Oxford > The Dyson Perrins Laboratory
Houssier, Claude ; University of Oxford > The Dyson Perrins Laboratory
Prendergast, Franklin G.; Mayo Foundation > Department of Biochemistry and Molecular Biology
Van Beeumen, Jozef; Rijksuniversiteit-Gent > Laboratorium voor Microbiologie en Microbiele Genetica
Ghuysen, Jean-Marie ; Université de Liège - ULiège > Institut de Chimie > Laboratoire d'Enzymologie / Centre d'Ingenierie des Proteines/Laboratoire de Chimie Macromoleculaire et Chimie Physique,
Frère, Jean-Marie ; Université de Liège - ULiège > Institut de Chimie > Centre d'ingénierie des protéines
Language :
English
Title :
Importance of the Two Tryptophan Residues in the Streptomyces R61 Exocellular Dd-Peptidase
Publication date :
01 March 1992
Journal title :
Biochemical Journal
ISSN :
0264-6021
eISSN :
1470-8728
Publisher :
Portland Press, London, United Kingdom
Volume :
282
Issue :
Pt 2
Pages :
361-367
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
FRSM - Fonds de la Recherche Scientifique Médicale [BE] F.R.S.-FNRS - Fonds de la Recherche Scientifique [BE] IRSIA - Intercommunale du Réseau Social d'Insertion et d'Accueil [BE]