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Understanding the molecular determinants of substrate and inhibitor specificities in the Carbapenemase KPC-2: exploring the roles of Arg220 and Glu276.

ABSTRACT: ?-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of ?-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A ?-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with ?-lactams and ?-lactamase inhibitors. To better understand the contribution of these residues to the catalytic process, we explored the roles of R220 and E276 in KPC-2, a class A ?-lactamase that inactivates carbapenems and ?-lactamase inhibitors. Our study reveals that substitutions at R220 of KPC-2 selectively impact catalytic activity toward substrates (50% or greater reduction in k(cat)/K(m)). In addition, we find that residue 220 is central to the mechanism of ?-lactamase inhibition/inactivation. Among the variants tested at Ambler position 220, the R220K enzyme is relatively "inhibitor susceptible" (K(i) of 14 ± 1 ?M for clavulanic acid versus K(i) of 25 ± 2 ?M for KPC-2). Specifically, the R220K enzyme is impaired in its ability to hydrolyze clavulanic acid compared to KPC-2. In contrast, the R220M substitution enzyme demonstrates increased K(m) values for ?-lactamase inhibitors (>100 ?M for clavulanic acid versus 25 ± 3 ?M for the wild type [WT]), which results in inhibitor resistance. Unlike other class A ?-lactamases (i.e., SHV-1 and TEM-1), the amino acid present at residue 276 plays a structural rather than kinetic role with substrates or inhibitors. To rationalize these findings, we constructed molecular models of clavulanic acid docked into the active sites of KPC-2 and the "relatively" clavulanic acid-susceptible R220K variant. These models suggest that a major 3.5-Å shift occurs of residue E276 in the R220K variant toward the active S70 site. We anticipate that this shift alters the shape of the active site and the positions of two key water molecules. Modeling also suggests that residue 276 may assist with the positioning of the substrate and inhibitor in the active site. These biochemical and molecular modeling insights bring us one step closer to understanding important structure-activity relationships that define the catalytic and inhibitor-resistant profile of KPC-2 and can assist the design of novel compounds.

SUBMITTER: Papp-Wallace KM

PROVIDER: S-EPMC3421566 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Understanding the molecular determinants of substrate and inhibitor specificities in the Carbapenemase KPC-2: exploring the roles of Arg220 and Glu276.

Papp-Wallace Krisztina M KM Taracila Magdalena A MA Smith Kerri M KM Xu Yan Y Bonomo Robert A RA

Antimicrobial agents and chemotherapy 20120611 8

β-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of β-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A β-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with β-lactams and β-lactamase inhibitors. To better understand ...[more]

PMID: 22687511

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Project description:Carbapenem-resistant Enterobacteriaceae (CRE) have emerged as major causes of health care-associated infections worldwide. This diverse collection of organisms with various resistance mechanisms is associated with increased lengths of hospitalization, costs of care, morbidity, and mortality. The global spread of CRE has largely been attributed to dissemination of a dominant strain of Klebsiella pneumoniae producing a serine β-lactamase, termed K. pneumoniae carbapenemase (KPC). Here we report an outbreak of KPC-producing CRE infections in which the degree of horizontal transmission between strains and species of a promiscuous plasmid is unprecedented. Sixteen isolates, comprising 11 unique strains, 6 species, and 4 genera of bacteria, were obtained from 14 patients over the first 8 months of the outbreak. Of the 11 unique strains, 9 harbored the same highly promiscuous plasmid carrying the KPC gene bla(KPC). The remaining strains harbored distinct bla(KPC) plasmids, one of which was carried in a strain of Klebsiella oxytoca coisolated from the index patient and the other generated from transposition of the bla(KPC) element Tn4401. All isolates could be genetically traced to the index patient. Molecular epidemiological investigation of the outbreak was aided by the adaptation of nested arbitrary PCR (ARB-PCR) for rapid plasmid identification. This detailed molecular genetic analysis, combined with traditional epidemiological investigation, provides insights into the highly fluid dynamics of drug resistance transmission during the outbreak. IMPORTANCE The ease of horizontal transmission of carbapenemase resistance plasmids across strains, species, and genera of bacteria observed in this study has several important public health and epidemiological implications. First, it has the potential to promote dissemination of carbapenem resistance to new populations of Enterobacteriaceae, including organisms of low virulence, leading to the establishment of reservoirs of carbapenem resistance genes in patients and/or the environment and of high virulence, raising the specter of untreatable community-associated infections. Second, recognition of plasmid-mediated outbreaks, such as those described here, is problematic because analysis of resistance plasmids from clinical isolates is laborious and technically challenging. Adaptation of nested arbitrary PCR (ARB-PCR) to investigate the plasmid outbreak facilitated our investigation, and the method may be broadly applicable to other outbreaks due to other conserved mobile genetic elements. Whether infection control measures that focus on preventing transmission of drug-resistant clones are effective in controlling dissemination of these elements is unknown.

Dataset Information

Understanding the molecular determinants of substrate and inhibitor specificities in the Carbapenemase KPC-2: exploring the roles of Arg220 and Glu276.

Publications

Understanding the molecular determinants of substrate and inhibitor specificities in the Carbapenemase KPC-2: exploring the roles of Arg220 and Glu276.

Similar Datasets

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