β-Lactam antibiotic targets and resistance mechanisms: from covalent inhibitors to substrates

Abstract

The β-lactams are the most widely used antibacterial agents worldwide. These antibiotics, a group that includes the penicillins and cephalosporins, are covalent inhibitors that target bacterial penicillin-binding proteins and disrupt peptidoglycan synthesis. Bacteria can achieve resistance to β-lactams in several ways, including the production of serine β-lactamase enzymes. While β-lactams also covalently interact with serine β-lactamases, these enzymes are capable of deacylating this complex, treating the antibiotic as a substrate. In this tutorial-style review, we provide an overview of the β-lactam antibiotics, focusing on their covalent interactions with their target proteins and resistance mechanisms. We begin by describing the structurally diverse range of β-lactam antibiotics and β-lactamase inhibitors that are currently used as therapeutics. Then, we introduce the penicillin-binding proteins, describing their functions and structures, and highlighting their interactions with β-lactam antibiotics. We next describe the classes of serine β-lactamases, exploring some of the mechanisms by which they achieve the ability to degrade β-lactams. Finally, we introduce the l,d-transpeptidases, a group of bacterial enzymes involved in peptidoglycan synthesis which are also targeted by β-lactam antibiotics. Although resistance mechanisms are now prevalent for all antibiotics in this class, past successes in antibiotic development have at least delayed this onset of resistance. The β-lactams continue to be an essential tool for the treatment of infectious disease, and recent advances (e.g., β-lactamase inhibitor development) will continue to support their future use.

Fig. 1. β-Lactam antibiotics and β-lactamase inhibitors. Chemical structure of the β-lactam ring, alongside generic structures of the major β-lactam antibiotic subclasses and representatives from these subclasses. The β-lactam ring in these structures is shown in red. Numbering typically begins from the atom in the top position of the ring fused to the β-lactam (e.g., sulfur in penicillins). The structures of common β-lactam-based and non-β-lactam-based β-lactamase inhibitors (BLIs) are also shown.

Fig. 2. Mechanism, inhibition, and structures of PBP transpeptidases. (A) Scheme showing the role of PBP transpeptidases in the formation of 4 → 3 peptidoglycan cross-links. The PBP nucleophilic serine attacks a pentapeptide on the donor strand, forming a peptide–enzyme complex and releasing d-alanine. From the acceptor strand of peptidoglycan, a nucleophilic side chain (here, l-lysine) reacts with the peptide–enzyme complex, forming a cross-link and releasing the enzyme. (B) Scheme showing the acylation of the transpeptidase serine nucleophile with a penicillin antibiotic, yielding an acyl–enzyme complex. (C) View of the transpeptidase domain of PBP2a from Staphylococcus aureus (PBP 1MWT). The nucleophilic serine, Ser403, is represented as white sticks. α-Helices are shown in green, β-sheets in yellow, and loops in white. The N-terminal domains and the region between residues 364 and 391 are not displayed; the C-terminus is labeled. (D) View of the active site of PBP1b from Escherichia coli (PDB 3VMA). Conserved active site residues, including the nucleophilic serine Ser403, are shown as sticks, and are coloured according to local secondary structure. (E) View of the active site of the transpeptidase domain of PBP2a from S. aureus in complex with benzylpenicillin (PDB 1MWT). The nucleophilic serine, Ser403, is acylated with benzylpenicillin (shown as tan sticks).

Fig. 3. Overview of serine β-lactamase mechanism and structure of class A enzymes. (A) Reaction scheme showing the main steps in SBL catalysis. In the acylation step, the SBL nucleophilic serine residue attacks the substrate β-lactam ring, yielding an ester-linked acyl–enzyme complex. In the hydrolysis step, the nucleophilic attack of a water molecule onto the acyl–enzyme complex carbonyl occurs, hydrolyzing the antibiotic and releasing the serine. (B) View of a crystal structure of class A SBL TEM-1 (PDB 1M40). The nucleophilic serine, Ser70, is represented as white sticks. α-Helices are shown in green, β-sheets in yellow, and loops in white. The N- and C-termini are indicated. (C) View of the active site of class A SBL GES-1 in which the serine (Ser64; green sticks) is acylated with the carbapenem imipenem (tan sticks) (PDB 4GOG). Conserved active site residues are represented as sticks and are coloured according to local secondary structure elements.

Fig. 4. Structures of class C serine β-lactamases. (A) View of the crystal structure of class C SBL AmpC from Escherichia coli (PDB 1KE4). The nucleophilic serine, Ser70, is represented as white sticks. α-Helices are shown in green, β-sheets in yellow, and loops in white. The N- and C-termini are labeled. (B) View of the active site of AmpC (PDB 1IEL). Conserved active site residues are represented as sticks and are coloured according to local secondary structure elements.

Fig. 5. Structures of class D serine β-lactamases. (A) View of the crystal structure of class D SBL OXA-10 (PDB 1EWZ). The nucleophilic serine, Ser70, is represented as green sticks. α-Helices are shown in green, β-sheets in yellow, and loops in white. The N- and C-termini are indicated. (B) View of the active site of the complex derived from OXA-48 and the carbapenem imipenem (PDB 5QB4). The nucleophilic serine Ser70 is acylated by imipenem (tan sticks). The side chain amino group of Lys73 of the SXXK motif is in the form of a carbamate, and is indicated as KCX73. Conserved active site residues are represented as sticks, and are coloured according to local secondary structure.

Fig. 6. Structure and inhibition of l,d-transpeptidases. (A) View of the transpeptidase domain of Ldt_Mt2 (PDB 6RLG), displaying the residues from 250 to the C-terminus. The nucleophilic cysteine, Cys354, is shown as white sticks. α-Helices are shown in green, β-sheets in yellow, and loops in white. The two N-terminal immunoglobulin fold-related domains are not shown. (B) Reaction scheme showing the acylation of the Ldt nucleophilic cysteine by the carbapenem meropenem, forming an acyl–enzyme complex. (C) View of the active site of Ldt_Mt2 in which the nucleophilic cysteine, Cys354 (white sticks), is acylated with meropenem (tan sticks) (PDB 4GSU). General base His336 and selected hydrophobic active site residues are shown as sticks and are coloured according to local secondary structure. (D) Reaction scheme showing the C5–C6 fragmentation of penicillin-derived and penem-derived acyl-enzyme complexes by Ldts.