The mechanism of action of DD-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 DD-peptidase.
The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results.
Wilkin
J M
JM
Centre d'Ingéniérie des protéines, Université de Liège, Belgium.
Dubus
A
A
Joris
B
B
Frère
J M
JM
D000215:Acylation; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004795:Enzyme Stability; D006868:Hydrolysis; D007700:Kinetics; D007769:Lactams; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics
D000900:Anti-Bacterial Agents; D007769:Lactams; D013912:Threonine; D002267:Muramoylpentapeptide Carboxypeptidase