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

Penicillin-binding protein 5 (PBP 5) of Escherichia coli functions as a D-a lanine carboxypeptidase, cleaving the C-terminal D-alanine residue from cel l 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 activi ty (deacylation), with only minor effects on acylation, and promotes accumu lation of a covalent complex with peptide substrates, To gain further insig ht into the catalytic mechanism of PBP 5, we determined the three-dimension al 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 penici llin 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 Ome ga loop of beta -lactamases. In beta -lactamases, the Omega loop contains t wo amino acids involved in catalyzing deacylation, This similarity may expl ain the high beta -lactamase activity of wild-type PBP 5. Because of the lo w rate of deacylation of the G105D mutant, visualization of peptide substra tes bound to the active site may be possible.