Crystal Structure of a Deacylation-defective Mutant of Penicillin-binding Protein 5 at 2.3-Å Resolution

Christopher Davies,Stephen W White,Robert A Nicholas

doi:10.1074/jbc.m004471200

Abstract

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 beta-lactam antibiotics; however, PBP 5 is distinguished by its high rate of deacylation of the acyl-enzyme complex (t(12) approximately 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 A 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.

Highlights

Penicillin and other ␤-lactam antibiotics exert their lethal effect by inhibiting the proteins that synthesize bacterial cell wall peptidoglycan [1]
A recent study utilizing strains with multiple deletions of low molecular mass PBPs revealed a role for Penicillin-binding protein 5 (PBP 5) in the proper synthesis of peptidoglycan [8]
The structure of sPBP 5Ј was determined by multiple isomorphous replacement (MIR) with anomalous scattering

Summary

Introduction

Penicillin and other ␤-lactam antibiotics exert their lethal effect by inhibiting the proteins that synthesize bacterial cell wall peptidoglycan [1]. These proteins, known as penicillinbinding proteins or PBPs, utilize lipid-linked disaccharide peptide substrates to catalyze both the polymerization of glycan chains (transglycosylation) and cross-linking of peptide chains (transpeptidation) during cell wall synthesis In the latter reaction, a serine residue on the PBP reacts with the acyl-D-Ala-D-Ala C terminus of the peptide chain to form a transient acyl-enzyme complex, releasing the C-terminal Dalanine residue. The atomic coordinates and structure factors (code 1hd8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) Another peptide chain to form a cross-link, which is crucial to the integrity and rigidity of the cell wall. These motifs are the SXXK tetrad containing the active site serine residue, the (S/Y)XN triad, and the KT(S)G triad

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Conclusion