Structural Basis for Clinical Longevity of Carbapenem Antibiotics in the Face of Challenge by the Common Class A β-Lactamases from the Antibiotic-Resistant Bacteria

Abstract

Bacteria resistant to antibiotics are being selected in a relatively short time, and cases of infections resistant to treatment by all known antibiotics are being identified at alarming rates. The primary mechanism for resistance to -lactam antibiotics is the catalytic function of -lactamases. However, imipenem (a -lactam) resists the action of most -lactamases and is virtually the last effective agent against the vancomycin-resistant Gram-positive bacteria, as well as against multiple antibiotic-resistant Gram-negative organisms. Here, we report the crystal structure, to 1.8 Å resolution, of an acyl-enzyme intermediate for imipenem bound to the TEM-1 -lactamase from Escherichia coli, the parent enzyme of 67 clinical variants. The structure indicates an unprecedented conformational change for the complex which accounts for the ability of this antibiotic to resist hydrolytic deactivation by -lactamases. Computational molecular dynamics underscored the importance of the motion of the acyl-enzyme intermediate, which may be a general feature for catalysis by these enzymes.

The High Resolution Crystal Structure for Class A β-Lactamase PER-1 Reveals the Bases for Its Increase in Breadth of Activity

“…Trp105 (an aromatic residue in most class A enzymes, Figure 4) has the same location as Tyr105 in TEM. In this protein, the aromatic ring is at van der Waals distance to the bound substrate in the crystal structures of enzyme-ligand complexes (52)(53)(54).…”

Exploring the potential impact of an expanded genetic code on protein function

“…Whether these interactions lower the free energy of activation by stabilizing the tetrahedral transition state or by introducing strain into the ground-state complex cannot be determined at present. However, the X-ray crystal structure of boronic acid transition-state analogs bound to the enzyme do show Tyr-105 in the same minus conformation that is present in the cephalexin acyl-enzyme intermediate of the Val-216-AcrF mutant, and the mutation does not show any significant effect on substrate K M (23,41,42). A comparison of the structures of the mutant and WT enzyme reveals no significant change in the position of the catalytic water molecule, supporting the notion that the Val-216-AcrF mutation primarily affects the first step of the enzymatic hydrolysis reaction.…”

Understanding the Structural Mechanisms of Antibiotic Resistance Sets the Platform for New Discovery

“…β-lactam antibiotic structures and imipenem, the first clinically available carbapenem antibiotic [13]. Crystal structures of imipenem bound to (B) TEM-1 (PDB: 1BT5, magenta) and (C) AmpC (PDB: 1LL5, blue) β-lactamases [14,15]. β-lactam antibiotics target the transpeptidase domain of penicillin-binding proteins (PBPs) involved in bacterial cell wall synthesis.…”

“…As β-lactamases have a high degree of structural homology to the PBPs, β-lactam antibiotics exhibit high affinity for these enzymes. β-lactams bind Class A β-lactamases and the nucleophilic active site serine acylates the carboxylate on the β-lactam ring via a ring opening reaction [15]. Once acylated, an active site water molecule hydrolyzes the acyl bond and the cleaved, deactivated β-lactam antibiotic is released from the active site [15].…”

“…β-lactams bind Class A β-lactamases and the nucleophilic active site serine acylates the carboxylate on the β-lactam ring via a ring opening reaction [15]. Once acylated, an active site water molecule hydrolyzes the acyl bond and the cleaved, deactivated β-lactam antibiotic is released from the active site [15]. Extended-spectrum β-lactamases (ESBLs), mainly from Class A, arose from early β-lactam use and are active in certain Gram-negative bacteria.…”