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.
abstract: 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 thiolester 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 Phe58----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.
authors.affiliation: Centre d'Ingénierie des Protéines, Université de Liège, Belgium.
authors.forename: A M
authors.initials: AM
authors.lastname: Hadonou
authors.forename: J M
authors.initials: JM
authors.lastname: Wilkin
authors.forename: L
authors.initials: L
authors.lastname: Varetto
authors.forename: B
authors.initials: B
authors.lastname: Joris
authors.forename: J
authors.initials: J
authors.lastname: Lamotte-Brasseur
authors.forename: D
authors.initials: D
authors.lastname: Klein
authors.forename: C
authors.initials: C
authors.lastname: Duez
authors.forename: J M
authors.initials: JM
authors.lastname: Ghuysen
authors.forename: J M
authors.initials: JM
authors.lastname: Frère
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D007700:Kinetics; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D009838:Oligodeoxyribonucleotides; D002267:Muramoylpentapeptide Carboxypeptidase; D001618:beta-Lactamases