6xj8

KPC-2 N170A mutant bound to hydrolyzed imipenem at 2.05 A

spinqualitylabelsall models 6xj8, resolution 2.05Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 6xj8 is a 1 chain structure with sequence from Klebsiella pneumoniae. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.05Å Ligands: 8YF Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function BLKPC_KLEPN Hydrolyzes carbapenems, penicillins, cephalosporins and monobactams with varying efficiency. Publication Abstract from PubMed Serine active-site beta-lactamases hydrolyze beta-lactam antibiotics through formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most beta-lactamases due to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared to the common TEM-1 beta-lactamase. Further, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase beta-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6alpha-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 beta-lactamase. KPC-2 beta-lactamase Enables Carbapenem Antibiotic Resistance Through Fast Deacylation of the Covalent Intermediate.,Mehta SC, Furey IM, Pemberton OA, Boragine DM, Chen Y, Palzkill T J Biol Chem. 2020 Dec 3. pii: RA120.015050. doi: 10.1074/jbc.RA120.015050. PMID:33273017 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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