8akm
Acyl-enzyme complex of ertapenem bound to deacylation mutant KPC-2 (E166Q)
Structural highlights
FunctionBLKPC_KLEPN Hydrolyzes carbapenems, penicillins, cephalosporins and monobactams with varying efficiency. Publication Abstract from PubMedKPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-beta-lactamase (SBL) responsible for extensive beta-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate beta-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent beta-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 A) 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 Omega-loop (residues 165-170) negatively correlates with antibiotic turnover rates (k(cat)), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different beta-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Delta1-(2R) imine rather than the Delta2 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 Delta1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Delta2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Delta2, rather than the Delta1-(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 Delta2 enamine-derived oxyanion. Taken together, our data show how the flexible Omega-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Delta2-enamine acyl-enzyme tautomer. Tautomer-Specific Deacylation and Omega-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 beta-Lactamase.,Tooke CL, Hinchliffe P, Beer M, Zinovjev K, Colenso CK, Schofield CJ, Mulholland AJ, Spencer J J Am Chem Soc. 2023 Apr 5;145(13):7166-7180. doi: 10.1021/jacs.2c12123. Epub 2023 , Mar 27. PMID:36972204[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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