Crystal structure of a deacylation-defective mutant of penicillin-binding protein 5 at 2.3-Å resolution

Penicillin-binding protein 5 (PBP 5) of Escherichia coli functions as a D-alanine carboxypeptidase, cleaving the C-terminal D-alanine residue from cell wall peptides, Like all PBPs, PBP 5 forms a covalent acyl-enzyme complex with p-lactam antibiotics; however, PBP 5 is distinguished by its high rate of deacylation of the acyl-enzyme complex (t(1/2) similar to 9 min). A Gly-105 --> Asp mutation in PBP 5 markedly impairs this beta -lactamase activity (deacylation), with only minor effects on acylation, and promotes accumulation of a covalent complex with peptide substrates, To gain further insight into the catalytic mechanism of PBP 5, we determined the three-dimensional structure of the G105D mutant form of soluble PBP 5 (termed sPBP 5') at 2.3 Angstrom resolution. The structure is composed of two domains, a penicillin binding domain with a striking similarity to Class A beta -lactamases (TEM-1-like) and a domain of unknown function. In addition, the penicillin-binding domain contains an active site loop spatially equivalent to the Omega loop of beta -lactamases. In beta -lactamases, the Omega loop contains two amino acids involved in catalyzing deacylation, This similarity may explain the high beta -lactamase activity of wild-type PBP 5. Because of the low rate of deacylation of the G105D mutant, visualization of peptide substrates bound to the active site may be possible.