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.
Importance of the two tryptophan residues in the Streptomyces R61 exocellular dd-peptidase
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C Bourguignon-Bellefroid, J M Wilkin, B Joris, R T Aplin, C Houssier, F G Prendergast, J Van Beeumen, J M Ghuysen, J M Frère; Importance of the two tryptophan residues in the Streptomyces R61 exocellular dd-peptidase. Biochem J 1 March 1992; 282 (2): 361–367. doi: https://doi.org/10.1042/bj2820361
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