The bacterial D-alanyl-D-alanine carboxypeptidase/transpeptidases (DD-peptidases) are penicillin-binding proteins (PBPs) that are responsible for cross-linking peptidoglycan polymers in the final step of bacterial cell wall biosynthesis. β-Lactam antibiotics, the primary treatments of bacterial infections, kill bacteria because they inhibit the DD-peptidases and disrupt cell wall synthesis. The future of β-lactams as effective chemotherapeutic agents is threatened by β-lactamases. These bacterial enzymes inactivate β-lactam antibiotics, rendering the host cell resistant to the bactericidal effects of the drugs. Nevertheless, the bacterial DD-peptidases remain attractive targets for antibiotic compounds. Structural studies can contribute in efforts to develop new, more effective antibiotics by providing detailed information about the DD-peptidase mechanism and the interactions of these enzymes with substrates and inhibitors. This work examines the X-ray crystallographic structures of a model DD-peptidase, the Streptomyces strain R61 DD-peptidase, in complexes with a number of inhibitors. The 1.1Å structure of the complex between the R61 enzyme and a phosphonate inhibitor gives new insight into the catalytic mechanism of this important class of enzyme by mimicking a tetrahedral transition state. We investigate ways to revitalize β-lactam antibiotics by looking at two novel compounds having an R61-specific peptidic side chain attached to either a penicillin or cephalosporin nucleus. In addition to two covalent β-lactamoyl enzyme complexes, we also trapped a non-covalent complex between the enzyme and the peptidyl penicillin. The covalent structures provide good analogs of the acyl enzyme with normal peptide substrates, completing the crystallographic imaging of the DD-peptidase mechanism. The non-covalent enzyme-inhibitor structure shows how the enzyme interacts with the β-lactam prior to inhibition. Finally, structures of complexes with bicyclic and monocyclic phosphates investigate the mode of action of this entirely novel class of DD-peptidase/β-lactamase inhibitor. The structures show that while the two inhibitors bind in nearly identical conformations, the phosphoryl enzyme complex formed with the monocyclic compound is significantly more stable than that formed with the bicyclic phosphate. This difference in the relative stabilities of the phosphoryl enzymes explains differences in the actions of these two compounds. Together, these structures will aid in future efforts to develop and improve DD-peptidase inhibitors.
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