Tautomer-specific deacylation and Ω-loop flexibility explain the carbapenem-hydrolyzing broad-spectrum activity of the KPC-2 β-lactamase

DOI: 10.1021/jacs.2c12123

Authors: Catherine L. Tooke (University of Bristol) , Philip Hinchliffe (University of Bristol) , Michael Beer (University of Bristol) , Kirill Zinovjev (University of Bristol) , Charlotte K. Colenso (University of Bristol) , Christopher J. Schofield (University of Oxford) , Adrian J. Mulholland (University of Bristol) , James Spencer (University of Bristol)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of The American Chemical Society

State: Published (Approved)
Published: March 2023
Diamond Proposal Number(s): 172122 , 23269

Abstract: KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-β-lactamase (SBL) responsible for extensive β-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate β-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent β-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25–1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165–170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different β-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.

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: 31/03/2023 09:10

Documents:
jacs.2c12123.pdf

Discipline Tags:

Pathogens Antibiotic Resistance Infectious Diseases Health & Wellbeing Biochemistry Chemistry Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)