Re‐examining the role of Lys67 in class C β‐lactamase catalysis

Abstract

Lys67 is essential for the hydrolysis reaction mediated by class C b-lactamases. Its exact catalytic role lies at the center of several different proposed reaction mechanisms, particularly for the deacylation step, and has been intensely debated. Whereas a conjugate base hypothesis postulates that a neutral Lys67 and Tyr150 act together to deprotonate the deacylating water, previous experiments on the K67R mutants of class C b-lactamases suggested that the role of Lys67 in deacylation is mainly electrostatic, with only a 2-to 3-fold decrease in the rate of the mutant vs the wild type enzyme. Using the Class C b-lactamase AmpC, we have reinvestigated the activity of this K67R mutant enzyme, using biochemical and structural studies. Both the rates of acylation and deacylation were affected in the AmpC K67R mutant, with a 61-fold decrease in k cat , the deacylation rate. We have determined the structure of the K67R mutant by X-ray crystallography both in apo and transition state-analog complexed forms, and observed only minimal conformational changes in the catalytic residues relative to the wild type. These results suggest that the arginine side chain is unable to play the same catalytic role as Lys67 in either the acylation or deacylation reactions catalyzed by AmpC. Therefore, the activity of this mutant can not be used to discredit the conjugate base hypothesis as previously concluded, although the reaction catalyzed by the K67R mutant itself likely proceeds by an alternative mechanism. Indeed, a manifold of mechanisms may contribute to hydrolysis in class C b-lactamases, depending on the enzyme (wt or mutant) and the substrate, explaining why different mutants and substrates seem to support different pathways. For the WT enzyme itself, the conjugate base mechanism may be well favored.

Avibactam and Class C β-Lactamases: Mechanism of Inhibition, Conservation of the Binding Pocket, and Implications for Resistance

“…Given the conservation of the avibactam binding pocket residues and the importance of these residues in ␤-lactam recognition and catalysis (25)(26)(27)(28)(29), we investigated whether any variations in these might compromise the inhibition potency of avibactam while still allowing hydrolysis of the ␤-lactam drug and thus result in resistance. Spontaneous resistance frequency experiments were carried out in several isolates carrying class C ␤-lactamases, with an initial focus on Enterobacteriaceae spp.…”

One ring to rule them all: Current trends in combating bacterial resistance to the β‐lactams

“…15 The class C cephalosporinase enzymes have a tyrosine rather that serine in motif ii, which is thought to be involved in protonation of the blactam nitrogen leaving group upon bond fission. 16 To overcome SBL resistance, three b-lactam based inhibitors have been introduced into clinical practice (sulbactam, clavulanic acid, and tazobactam). Sulbactam and tazobactam are penicillanic acid sulfones, 17 while clavulanic acid is a clavam secondary metabolite from Streptomyces clavuligerus originally isolated in the early 1970s.…”

Hydrolytic Mechanism of OXA-58 Enzyme, a Carbapenem-hydrolyzing Class D β-Lactamase from Acinetobacter baumannii

“…In classes A and C ␤-lactamases, the catalytic water molecule is anchored through a hydrogen bond network in an optimum position for nucleophilic attack on the carbonyl carbon of the acyl-enzyme species. The position of this water molecule is crystallographically conserved (31,36,41) unlike in class D enzymes (42)(43)(44). Perturbation of the hydrogen bonding network of this water molecule or the physical displacements of the water molecule by the steric bulk of the C-6 side chains of a ␤-lactam are possible mechanisms to render the ␤-lactamase deacylation-deficient (20,(45)(46)(47).…”