Article (Scientific journals)
Site-Directed Mutagenesis of the Actinomadura R39 DD-Peptidase
Zhao, GuoHua; Duez, Colette; Forceille, Christine et al.
1997In Biochemical Journal, 327 (2), p. 377-381
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Keywords :
Mutagenèse dirigée; R39 DD-peptidase
Abstract :
[en] The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive dd-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the 'SDN loop' by Ala or Gly significantly decreased the kcat/Km value for the peptide substrate, but only by a factor of 15 and had little effect on the other catalytic properties. Mutations of Asn-300 of the same loop and of Lys-410 of the KTG triad yielded very unstable proteins. However, the N300S mutant could be purified as a fusion protein with thioredoxin that exhibited decreased rates of acylation by the peptide substrate and various cephalosporins. Similar fusion proteins obtained with the N300A, K410H and K410N mutants were unstable and their catalytic and penicillin-binding properties were very strongly affected. In transpeptidation reactions, the presence of the acceptor influenced the kcat/Km values, which suggested a catalytic pathway more complex than a simple partition of the acyl-enzyme between hydrolysis and aminolysis. These results are compared with those obtained with two other penicillin-sensitive enzymes, the Streptomyces R61 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5.
Research center :
CIP - Centre d'Ingénierie des Protéines - ULiège
Disciplines :
Biochemistry, biophysics & molecular biology
Microbiology
Author, co-author :
Zhao, GuoHua;  Université de Liège - ULiège > Centre d'ingénierie des protéines > Laboratoire d'Enzymologie
Duez, Colette ;  Université de Liège - ULiège > Centre d'ingénierie des protéines > Laboratoire d'Enzymologie
Forceille, Christine;  Université de Liège - ULiège > Laboratoire de référence SIDA
Rhazi, Noureddine ;  Université de Liège - ULiège > Centre d'ingénierie des protéines > Laboratoire d'Enzymologie
Klein, Daniel;  Université de Liège - ULiège > Centre d'ingénierie des protéines > Laboratoire d'Enzymologie
Ghuysen, Jean-Marie ;  Université de Liège - ULiège > Centre d'ingénierie des protéines > Laboratoire d'Enzymologie
Frère, Jean-Marie ;  Université de Liège - ULiège > Centre d'Ingénierie des Protéines > Laboratoire d'Enzymologie
Lepage, Sophie;  Université de Liège - ULiège > Centre d'ingénierie des protéines > Laboratoire d'Enzymologie
Language :
English
Title :
Site-Directed Mutagenesis of the Actinomadura R39 DD-Peptidase
Publication date :
15 October 1997
Journal title :
Biochemical Journal
ISSN :
0264-6021
eISSN :
1470-8728
Publisher :
Portland Press, London, United Kingdom
Volume :
327
Issue :
2
Pages :
377-381
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
FRSM - Fonds de la Recherche Scientifique Médicale [BE]
Available on ORBi :
since 09 January 2009

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