Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance

Abstract

CTX-M β-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-μs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restricting the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories.

Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl–enzyme conformational dynamics to extend antibiotic resistance

“…The first of these is proposed to increase conformational heterogeneity of the Ω-loop to better accommodate ceftazidime (60), while E104K/E240K may participate in direct interactions with ceftazidime (59). In the CTX-M ESBL family, where the parent hydrolyzes cefotaxime efficiently but ceftazidime poorly, turnover of the latter is moderately improved by point mutations such as P167S or D240G that are proposed to increase Ω-loop flexibility to a more "open" conformation, improving accommodation of ceftazidime while E166 remains in a deacylation-competent orientation (61). This contrasts with KPC, in which our high resolution crystal structures and MD simulations indicate greatly enhanced ceftazidime turnover by KPC-4 (compared to KPC-2) is instead associated with reduced flexibility, or increased stability, of the acylenzyme Ω-loop, constraining E166 in an orientation compatible with productive deacylation.…”

“…However, despite the structural similarity between class A β-lactamases, available data indicate that populated acyl–enzyme conformations, and their associated hydrolytic activities, vary between different SBLs. This is exemplified by the apparently opposing effects of increased Ω loop flexibility upon ceftazidime hydrolysis in the CTX-M ( 36 , 60 ) and KPC enzyme families. These differences likely reflect differing active-site structures and associated activity spectra of the progenitor enzymes as well as differences in stability/dynamics of the overall enzyme structure.…”

Local interactions with the Glu166 base and the conformation of an active site loop play key roles in carbapenem hydrolysis by the KPC-2 β-lactamase

“…Active site loop structures have been shown to play an important role in β-lactamase substrate specificity ( 8 , 45 ). It has been extensively documented that changes in the 164 to 179 omega loop, which contributes to the floor of the active site and contains Glu166, result in altered substrate specificity of class A β-lactamases ( 8 , 45 , 46 , 47 , 48 , 49 , 50 , 51 ). Changes in the conformation of the 103 to 106 loop are also associated with changes in substrate specificity ( 41 ).…”

Defense by duplication: The relation between phenotypic glyphosate resistance and EPSPS gene copy number variation inAmaranthus palmeri

“…Negative epistatic interactions with EPSPS CNV have the potential to decrease the phenotypic effect of EPSPS copy number. In studies of microbial resistance negative epistasis has been frequently detected (Brown et al, 2020;Lukacisinova, Fernando, & was not certified by peer review) is the author/funder. All rights reserved.…”