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

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
Diamond Keywords: Bacteria; Enzymes
Subject Areas:
Biology and Bio-materials,
Chemistry,
Medicine
Instruments:
I04-Macromolecular Crystallography
,
I24-Microfocus Macromolecular Crystallography
Other Facilities: BL13- XALOC at ALBA
Added On:
09/12/2020 11:17
Discipline Tags:
Pathogens
Antibiotic Resistance
Infectious Diseases
Health & Wellbeing
Biochemistry
Chemistry
Structural biology
Drug Discovery
Life Sciences & Biotech
Technical Tags:
Diffraction
Macromolecular Crystallography (MX)