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

• Published in: The Journal of biological chemistry 2021-01, Vol.296, p.100126, Article 100126
• Main Authors: Tooke, Catherine L., Hinchliffe, Philip, Bonomo, Robert A., Schofield, Christopher J., Mulholland, Adrian J., Spencer, James
• Format: Article
• Language: English
• Subjects: Acylation; acyl–enzyme; Anti-Bacterial Agents - pharmacology; antibiotic resistance; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; beta-Lactamases - chemistry; beta-Lactamases - genetics; beta-Lactamases - metabolism; Catalytic Domain; ceftazidime; Ceftazidime - pharmacology; crystal structure; Drug Resistance, Microbial; enzyme catalysis; Hydrolysis; Kinetics; Klebsiella pneumoniae - drug effects; Klebsiella pneumoniae - enzymology; Klebsiella pneumoniae - genetics; Models, Molecular; molecular dynamics; Mutagenesis, Site-Directed; Mutation; Protein Conformation; serine β-lactamase; structure–function; β-lactam
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA120.016461

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), for example, 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 that KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (kcat), rather than altered KM values. 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, which 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.