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

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 expandedspectrum 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 , 240, and 270 loops, which border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acylenzyme and are not observed in the KPC-2:cefotaxime acylenzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the 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 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.