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

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 20000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe58 --> Leu, Tyr9O --> 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.