Reference : Site-Directed Mutagenesis of the Actinomadura R39 DD-Peptidase
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
Life sciences : Microbiology
http://hdl.handle.net/2268/3975
Site-Directed Mutagenesis of the Actinomadura R39 DD-Peptidase
English
Zhao, GuoHua [Université de Liège - ULg > > Centre d'ingénierie des protéines > Laboratoire d'Enzymologie > >]
Duez, Colette mailto [Université de Liège - ULg > > Centre d'ingénierie des protéines >Laboratoire d'Enzymologie > >]
Forceille, Christine [Université de Liège - ULg > > > Laboratoire de référence SIDA > > >]
Rhazi, Noureddine mailto [Université de Liège - ULg > > Centre d'ingénierie des protéines > Laboratoire d'Enzymologie > >]
Klein, Daniel [Université de Liège - ULg > > Centre d'ingénierie des protéines > Laboratoire d'Enzymologie > >]
Ghuysen, Jean-Marie [Université de Liège - ULg > > Centre d'ingénierie des protéines > Laboratoire d'Enzymologie > >]
Frère, Jean-Marie [Université de Liège - ULg > > Centre d'Ingénierie des Protéines > Laboratoire d'Enzymologie > >]
Lepage, Sophie [Université de Liège - ULg > > Centre d'ingénierie des protéines > Laboratoire d'Enzymologie > >]
15-Oct-1997
Biochemical Journal
Portland Press
327
2
377-381
Yes (verified by ORBi)
International
0264-6021
1470-8728
London
United Kingdom
[fr] Mutagenèse dirigée ; R39 DD-peptidase
[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.
Centre d'Ingénierie des Protéines - CIP
Fonds de la Recherche Scientifique Médicale - FRSM
Researchers ; Professionals
http://hdl.handle.net/2268/3975

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