Abstract
Class A serine β-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize β-lactams via a hydrolytically labile covalent acyl–enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent β-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), for example, cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show that KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (kcat), rather than altered KM values. High-resolution crystal structures of ESOC acyl–enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω, 240, and 270 loops, which border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω loop, adopts two distinct conformations in KPC-2, either pointing “in” or “out” of the active site; with only the "in" form compatible with deacylation. The "out" conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl–enzyme intermediate.
Highlights
Increasing antimicrobial resistance threatens global public health [1], exemplified by resistance to β-lactams, the most prescribed antibiotics worldwide [2]
A substrate resembling ceftazidime, but with a smaller C7 group (Fig. 1), KM values are lower than those for ceftazidime for both Klebsiella pneumoniae carbapenemase (KPC)-2 and KPC-4 (200 and 190 μM, respectively) and kcat values substantially faster (76 and 262 s−1), resulting in KPC-4 ~fourfold more efficiently hydrolyzing cefotaxime than Klebsiella pneumoniae carbapenemase-2 (KPC-2)
We note that overall KM values for expandedspectrum oxyimino-cephalosporins (ESOCs) turnover (Table 1) reflect contributions from the microscopic rate constants for multiple individual steps along the hydrolysis pathway [40], and that in consequence, KM will be sensitive to changes in the structures and dynamics of transient species as well as those of the acyl–enzyme state observed here, our structural data suggest that the lower KM value for KPC-2–catalyzed cefotaxime, compared with ceftazidime, hydrolysis may in part reflect the lack of a requirement for significant active-site rearrangements in the cefotaxime acyl-enzyme
Summary
Increasing antimicrobial resistance threatens global public health [1], exemplified by resistance to β-lactams (e.g. carbapenems and cephalosporins), the most prescribed antibiotics worldwide [2]. We note that overall KM values for ESOC turnover (Table 1) reflect contributions from the microscopic rate constants for multiple individual steps along the hydrolysis pathway [40], and that in consequence, KM will be sensitive to changes in the structures and dynamics of transient species as well as those of the acyl–enzyme state observed here, our structural data suggest that the lower KM value for KPC-2–catalyzed cefotaxime, compared with ceftazidime, hydrolysis may in part reflect the lack of a requirement for significant active-site rearrangements in the cefotaxime acyl-enzyme.
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