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

DOI: 10.1074/jbc.RA120.016461 DOI Help

Authors: Catherine L. Tooke (University of Bristol) , Philip Hinchliffe (University of Bristol) , Robert A. Bonomo (Louis Stokes Cleveland VAMC) , Christopher J. Schofield (Oxford University) , Adrian J. Mulholland (University of Bristol) , James Spencer (University of Bristol)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: November 2020
Diamond Proposal Number(s): 172122 , 23269

Open Access Open Access

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), e.g. 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 KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (kcat), rather than binding (KM). 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, that 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.

Journal Keywords: Serine β-lactamase; β-lactam; ceftazidime; acyl-enzyme; enzyme catalysis; antibiotic resistance; X-ray crystallography; structure-function; molecular dynamics

Subject Areas: Biology and Bio-materials, Chemistry, Medicine

Instruments: I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Other Facilities: BL13- XALOC at ALBA

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