Crystallographic Snapshots of Class A β-Lactamase Catalysis Reveal Structural Changes That Facilitate β-Lactam Hydrolysis

Abstract

β-Lactamases confer resistance to β-lactam-based antibiotics. There is great interest in understanding their mechanisms to enable the development of β-lactamase-specific inhibitors. The mechanism of class A β-lactamases has been studied extensively, revealing Lys-73 and Glu-166 as general bases that assist the catalytic residue Ser-70. However, the specific roles of these two residues within the catalytic cycle remain not fully understood. To help resolve this, we first identified an E166H mutant that is functional but is kinetically slow. We then carried out time-resolved crystallographic study of a full cycle of the catalytic reaction. We obtained structures that represent apo, S*-acylation, andS*-deacylation states and analyzed the conformational changes of His-166. The "in" conformation in the apo structure allows His-166 to form a hydrogen bond with Lys-73. The unexpected "flipped-out" conformation of His-166 in the S*-acylation structure was further examined by molecular dynamics simulations, which suggested deprotonated Lys-73 serving as the general base for acylation. The "revert-in" conformation in theS*-deacylation structure aligns His-166 toward the water molecule that hydrolyzes the acyl adduct. Finally, when the acyl adduct is fully hydrolyzed, His-166 rotates back to the "in" conformation of the apo-state, restoring the Lys-73/His-166 interaction. Using His-166 as surrogate, our study identifies distinct conformational changes within the active site during catalysis. We suggest that the native Glu-166 executes similar changes in a less constricted way. Taken together, this structural series improves our understanding of β-lactam hydrolysis in this important class of enzymes.

Structural Insights into the Inhibition of the Extended-Spectrum β-Lactamase PER-2 by Avibactam

“…Overall, our structures reveal that Ser70 is primed by deprotonation for nucleophilic attack on the AVI C-7 atom. It is still uncertain if this deprotonation is driven by Lys73 or Glu166 via a catalytic water that bridges both Glu166 and Ser70 (17)(18)(19)(20)(21)(22). Using both highresolution structural studies and molecular dynamics simulations, both Lys73 and Ser130 are also believed to participate in a proton shuttle resulting in the protonation of the AVI N-6 in the acylation step (15,23).…”

β-Lactamase of Mycobacterium tuberculosis Shows Dynamics in the Active Site That Increase upon Inhibitor Binding

“…As has been suggested before (e.g. (56,58,59)), the Lys73 sidechain may need to be in one conformation for efficient substrate binding and another for efficient product release. Thus, it seems possible that the two conformations observed in crystal structures of Toho-1 ( [57][58][59][60] and BlaC, (53) are the cause of the two forms that we observe in the NMR spectra of the BlaCclavulanic acid adduct.…”

Modified Penicillin Molecule with Carbapenem-Like Stereochemistry Specifically Inhibits Class C β-Lactamases

“…PenP is a class A ␤-lactamase from Bacillus licheniformis with over 80% sequence similarity to the clinically significant TEM-1. Our previous studies have also confirmed that the kinetic profile of PenP is nearly identical to that for TEM-1, showing a narrow substrate profile with more effective hydrolysis of only first-and second-generation penicillins and cephalosporins (19,20). P99, on the other hand, is an AmpC ␤-lactamase (also termed class C ␤-lactamase) from Enterobacter cloacae.…”