Characterization of the Global Stabilizing Substitution A77V and Its Role in the Evolution of CTX-M β-Lactamases

Abstract

The widespread use of oxyimino-cephalosporin antibiotics drives the evolution of the CTX-M family of ␤-lactamases that hydrolyze these drugs and confer antibiotic resistance. Clinically isolated CTX-M enzymes carrying the P167S or D240G active siteassociated adaptive mutation have a broadened substrate profile that includes the oxyimino-cephalosporin antibiotic ceftazidime. The D240G substitution is known to reduce the stability of CTX-M-14 ␤-lactamase, and the P167S substitution is shown here to also destabilize the enzyme. Proteins are marginally stable entities, and second-site mutations that stabilize the enzyme can offset a loss in stability caused by mutations that enhance enzyme activity. Therefore, the evolution of antibiotic resistance enzymes can be dependent on the acquisition of stabilizing mutations. The A77V substitution is present in CTX-M extendedspectrum ␤-lactamases (ESBLs) from a number of clinical isolates, suggesting that it may be important in the evolution of antibiotic resistance in this family of ␤-lactamases. In this study, the effects of the A77V substitution in the CTX-M-14 model enzyme were characterized with regard to the kinetic parameters for antibiotic hydrolysis as well as enzyme expression levels in vivo and protein stability in vitro. The A77V substitution has little effect on the kinetics of oxyimino-cephalosporin hydrolysis, but it stabilizes the CTX-M enzyme and compensates for the loss of stability resulting from the P167S and D240G mutations. The acquisition of global stabilizing mutations, such as A77V, is an important feature in ␤-lactamase evolution and a common mechanism in protein evolution.T he introduction of new antibiotics into clinical use fuels the evolution of enzymes causing drug resistance. These enzymes gain the ability to inactivate antibiotics more efficiently by acquiring point mutations in and around their active sites that increase catalytic activity (1). However, the introduction of new gain-offunction mutations within highly organized active sites often comes at a stability cost to the enzymes (2-6). Therefore, enzymes can absorb only a limited number of destabilizing mutations before they unfold and lose function (7). This limitation in the evolution of catalytic efficiency is often overcome by the acquisition of global stabilizing mutations that offset the incremental loss in stability due to the primary mutation. This compensation mechanism allows the enzymes to continue a mutational trajectory, resulting in increased resistance.The role of global stabilizing mutations in enzyme evolution has been widely studied (4,8,9). In recent decades, the selection of mutant ␤-lactamase enzymes with expanded substrate specificity has occurred for the plasmid-mediated TEM-and SHV-type ␤-lactamases to give rise to extended-spectrum ␤-lactamases (ESBLs) (10, 11). These enzymes arise due to mutations resulting in substitutions near the active site that increase hydrolysis of oxyimino-cephalosporins such as cefotaxime and ceftazidime. The substitutions that increa...

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation

“…The partial density for ceftazidime may be due to high mobility of these groups and could be related to the high K m observed for ceftazidime hydrolysis in the CTX-M-14 P167S enzyme. 11 …”

“…The CTX-M-14-pET28a plasmid was created by inserting the gene encoding wild-type CTX-M-14 into the pET28a plasmid using the Gibson Assembly Kit (New England BioLabs, Ipswich, MA) as previously described. 11 The CTX-M-14-pET28a plasmid was transformed into E. coli XL1-Blue [ recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac, [F9 proAB lacIq lacZΔM15, Tn10 (tetr)]] (Stratagene, Inc., La Jolla, CA) for construction of the CTX-M-14 mutants by site-directed mutagenesis. 20 The pET28a plasmids encoding the wild-type and mutant β-lactamases were transformed into the E. coli strain BL21 (DE3)( fhuA2[lon]ompTgal(λDE3) [dcm] Δ hsdSλDE3=λsBamHIo Δ EcoRI-B int::(lacI::PlacUV5::T7gene1) i21 Δ nin5 ) for protein expression and purification.…”

“…In particular, the D240G and P167S substitutions occur individually in multiple CTX-M subgroups where they enhance ceftazidime hydrolysis. 8–11 …”

“…The increased catalytic efficiency for ceftazidime hydrolysis by CTX-M-14 P167S, comes at a cost of stability and decreased ability to provide resistance against older β-lactam antibiotics. 8,11,13–15 The P167S mutation is located in the Ω-loop, which forms the bottom of the active site in CTX-M enzymes. 16,17 It is located adjacent to the Glu166 residue that is critical in activating the catalytic water molecule for deacylation.…”

Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity

“…The mutations that stabilize the hydrophobic core of NDM, particularly M154L, likely serve as global suppressors that enable the introduction of other mutations, which can add function at the price of structural destabilization. Global suppressing mutations have been previously noted in the evolution of serine β- [20][21][22]. For example, in the NDM variants, introduction of the E170K mutation decreases Tm by 1.7 ± 0.2 °C (Tm, NDM-17 -Tm, , but the stabilizing effect of the other two mutations found in NDM-17 more than compensates to yield an overall thermostability that is still greater than that of NDM-1.…”