6Z25

Acylenzyme complex of ceftazidime bound to deacylation mutant KPC-4 (E166Q)

Summary for 6Z25

• Entry DOI: 10.2210/pdb6z25/pdb
• Descriptor: Beta-lactamase, GLYCEROL, ACYLATED CEFTAZIDIME, ... (5 entities in total)
• Functional Keywords: unliganded beta-lactamase, 3-layer alpha-beta-alpha sandwich, part of the dd-peptidase and beta-lactamase superfamily., antimicrobial protein
• Biological source: Klebsiella pneumoniae
• Total number of polymer chains: 1
• Total formula weight: 32141.83

Authors

Tooke, C.L.,Hinchliffe, P.,Spencer, J. (deposition date: 2020-05-14, release date: 2020-12-23, Last modification date: 2024-10-23)

Primary citation

Tooke, C.L.,Hinchliffe, P.,Bonomo, R.A.,Schofield, C.J.,Mulholland, A.J.,Spencer, J.
Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance.
J.Biol.Chem., 296:100126-100126, 2020

PubMed Abstract: 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 (k), rather than altered K 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.

PubMed: 33257320
DOI: 10.1074/jbc.RA120.016461

Experimental method

X-RAY DIFFRACTION (1.24 Å)