Crystal Structures of Complexes between the R61 DD-peptidase and Peptidoglycan-mimetic β-Lactams: A Non-covalent Complex with a “Perfect Penicillin”

The bacterial d -alanyl- d -alanine transpeptidases (DD-peptidases) are the killing targets of β-lactams, the most important clinical defense against bacterial infections. However, due to the constant development of antibiotic-resistance mechanisms by bacteria, there is an ever-present need for new, more effective antimicrobial drugs. While enormous numbers of β-lactam compounds have been tested for antibiotic activity in over 50 years of research, the success of a β-lactam structure in terms of antibiotic activity remains unpredictable. Tipper and Strominger suggested long ago that β-lactams inhibit DD-peptidases because they mimic the d -alanyl- d -alanine motif of the peptidoglycan substrate of these enzymes. They also predicted that β-lactams having a peptidoglycan-mimetic side-chain might be better antibiotics than their non-specific counterparts, but decades of research have not provided any evidence for this. We have recently described two such novel β-lactams. The first is a penicillin having the glycyl- l -α-amino-e-pimelyl side-chain of Streptomyces strain R61 peptidoglycan, making it the “perfect penicillin” for this organism. The other is a cephalosporin with the same side-chain. Here, we describe the X-ray crystal structures of the perfect penicillin in non-covalent and covalent complexes with the Streptomyces R61 DD-peptidase. The structure of the non-covalent enzyme–inhibitor complex is the first such complex to be trapped crystallographically with a DD-peptidase. In addition, the covalent complex of the peptidyl-cephalosporin with the R61 DD-peptidase is described. Finally, two covalent complexes with the traditional β-lactams benzylpenicillin and cephalosporin C were determined for comparison with the peptidyl β-lactams. These structures, together with relevant kinetics data, support Tipper and Strominger's assertion that peptidoglycan-mimetic side-chains should improve β-lactams as inhibitors of DD-peptidases.