8AKJ

Acyl-enzyme complex of cephalothin bound to deacylation mutant KPC-2 (E166Q)

8AKJ の概要

• エントリーDOI: 10.2210/pdb8akj/pdb
• 分子名称: Carbapenem-hydrolyzing beta-lactamase KPC, SULFATE ION, GLYCEROL, ... (5 entities in total)
• 機能のキーワード: acyl-enzyme complex, antibiotic resistance, beta-lactamase, antibiotic, ligand, antimicrobial protein
• 由来する生物種: Klebsiella pneumoniae
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 31708.52

構造登録者

Tooke, C.L.,Hinchliffe, P.,Spencer, J. (登録日: 2022-07-29, 公開日: 2023-03-08, 最終更新日: 2024-02-07)

主引用文献

Tooke, C.L.,Hinchliffe, P.,Beer, M.,Zinovjev, K.,Colenso, C.K.,Schofield, C.J.,Mulholland, A.J.,Spencer, J.
Tautomer-Specific Deacylation and Omega-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 beta-Lactamase.
J.Am.Chem.Soc., 145:7166-7180, 2023

PubMed Abstract: KPC-2 ( 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 (), 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-(2) 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-(2) 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-(2) 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.

PubMed: 36972204
DOI: 10.1021/jacs.2c12123

実験手法

X-RAY DIFFRACTION (1.35 Å)