title: Importance of the two tryptophan residues in the Streptomyces R61 exocellular DD-peptidase.
abstract: 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.
authors.affiliation: Laboratoire d'Enzymologie, Université de Liège, Belgium.
authors.forename: C
authors.initials: C
authors.lastname: Bourguignon-Bellefroid
authors.forename: J M
authors.initials: JM
authors.lastname: Wilkin
authors.forename: B
authors.initials: B
authors.lastname: Joris
authors.forename: R T
authors.initials: RT
authors.lastname: Aplin
authors.forename: C
authors.initials: C
authors.lastname: Houssier
authors.forename: F G
authors.initials: FG
authors.lastname: Prendergast
authors.forename: J
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: J M
authors.initials: JM
authors.lastname: Ghuysen
authors.forename: J M
authors.initials: JM
authors.lastname: Frère
mesh_terms: D001483:Base Sequence; D001974:Bromosuccinimide / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010957:Plasmids; D013050:Spectrometry, Fluorescence; D013302:Streptomyces / Q000201:enzymology* / Q000254:growth & development; D014364:Tryptophan / Q000235:genetics* / Q000378:metabolism; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D014364:Tryptophan; D002267:Muramoylpentapeptide Carboxypeptidase; D001618:beta-Lactamases; D001974:Bromosuccinimide