Three factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys70

Abstract

The activity of class D β-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate-limiting step for the wild-type OXA-10 β-lactamase.

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

“…The kinetics of nitrocefin hydrolysis was not affected by bicarbonate concentration, as assessed by using UVspectrophotometer. Similar observations were made by Vercheval et al (26). Nitrocefin is intrinsically a chemically reactive molecule and as such partial loss or gaining of carbamylated species may not have an impact in the turnover rate of nitrocefin.…”

“…Hydrolysis progress curves for several ␤-lactams with OXA-10 displayed biphasic kinetics, which simplified to monophasic when buffers were supplemented with NaHCO 3 (the source of CO 2 ). These studies demonstrated that the biphasicity in kinetics was due to the decarbamylation of the lysine active site residue during the kinetics (26,27).…”

“…Similar observations have been made for OXA-1 and OXA-10 (25, 26). Vercheval et al (26) reported that a K70A mutation in OXA-10 reduced its catalytic efficiency by 4,250-fold (…”

“…In class C, activation of the serine may occur by either Lys-67 or Tyr-105 as the general base (34 -36). In class D ␤-lactamases, a carbamylated lysine serves as the general base in both steps (26,27,37). The steady state kinetic parameters of OXA-58 ⌬SP , determined in the presence or absence of CO 2 , establishes the role of CO 2 in the creation of the catalytic machinery of the active site.…”

Sensitive and Specific Phenotypic Assay for Metallo-β-Lactamase Detection in Enterobacteria by Use of a Moxalactam Disk Supplemented with EDTA

“…The moxalactam is an extended-spectrum cephamycin that possesses a methoxy group at position 7 in the ␤-lactam ring. Previous studies suggested that this compound was poorly inactivated by class A, C, and D carbapenemases (14,16,17,24), whereas it was well hydrolyzed by class B enzymes (21). To confirm this statement, we determined the MIC values of several extended-spectrum ␤-lactams, including moxalactam, for Escherichia coli DH10B (bla KPC-3 ), E. coli DH10B (bla NDM-1 ), E. coli DH10B (bla VIM-4 ), E. coli TOP10 (pCMY-2), and E. coli J53-2 (bla OXA-48 ) recombinant or transconjugant strains, which produced KPC-3, NDM-1, VIM-4, CMY-2, and OXA-48 enzymes, respectively (7,8,12,14).…”