Substrate Selectivity and a Novel Role in Inhibitor Discrimination by Residue 237 in the KPC-2 β-Lactamase

Papp-Wallace, Krisztina M.; Taracila, Magdalena A.; Hornick, John M.; Hujer, Andrea M.; Hujer, Kristine M.; Distler, Anne M.; Endimiani, Andrea; Bonomo, Robert A.

doi:10.1128/aac.00197-10

Cited by 48 publications

(58 citation statements)

References 60 publications

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“…Papp-Wallace et al previously observed this ''clavulanic acid-resistant, sulfone-susceptible'' phenotype with the variants of KPC-2 at position 237 expressed in E. coli. 14 We attribute this similar phenotype at Ambler position 105 to important differences in the reaction mechanisms of clavulanic acid and the sulfones with KPC-2 and its variants. For instance, clavulanic acid's partition ratio is higher than for the sulfones with the variants and K i s for clavulanic acid varying significantly more than for the sulbactam and tazobactam.…”

Section: Inhibitor Discriminationmentioning

confidence: 89%

“…11,14 Briefly, cells were grown in super optimal broth, pelleted, and frozen for 18 h at À20 C. Pellets were subjected to stringent periplasmic fractionation, and crude extracts were used for preparative isoelectric focusing. Preparative isoelectric focusing fractions were concentrated and further purified using Ä kta fast protein liquid chromatography.…”

Section: B-lactamase Purificationmentioning

confidence: 99%

“…11,14 The extinction coefficient used for doripenem: De ¼ À11,460 M À1 cm À1 at 299 nm. Briefly, each assay was completed in 10 mM PBS at pH 7.4 at room temperature with the enzyme (E) to substrate (S) concentrations set to establish pseudo first-order kinetics, where the concentration of enzyme is negligible [Eq.…”

Section: Kineticsmentioning

confidence: 99%

“…We previously assessed the role of Ambler position 13 Thr237 in KPC-2 and found that this residue is necessary for selected cephalosporinase and carbapenemase activity. 14 In addition, we discovered that Thr237 discriminates between clavulanic acid and the penicillin sulfone inhibitors (sulbactam and tazobactam). 14 In this study, we chose to examine the role of another important active site residue, Ambler position Trp105 in KPC-2 blactamase.…”

Section: Introductionmentioning

confidence: 99%

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Elucidating the role of Trp105 in the KPC‐2 β‐lactamase

et al. 2010

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The molecular basis of resistance to b-lactams and b-lactam-b-lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective b-lactam therapy. Recent crystal structures of KPC-2 and other class A b-lactamases suggest that Ambler position Trp105 may be of importance in binding b-lactam compounds. Based on this notion, we explored the role of residue Trp105 in KPC-2 by conducting site-saturation mutagenesis at this position. Escherichia coli DH10B cells expressing the Trp105Phe, -Tyr, -Asn, and -His KPC-2 variants possessed minimal inhibitory concentrations (MICs) similar to E. coli cells expressing wild type (WT) KPC-2. Interestingly, most of the variants showed increased MICs to ampicillin-clavulanic acid but not to ampicillin-sulbactam or piperacillin-tazobactam. To explain the biochemical basis of this behavior, four variants (Trp105Phe, -Asn, -Leu, and -Val) were studied in detail. Consistent with the MIC data, the Trp105Phe b-lactamase displayed improved catalytic efficiencies, k cat /K m , toward piperacillin, cephalothin, and nitrocefin, but slightly decreased k cat /K m toward cefotaxime and imipenem when compared to WT b-lactamase. The Trp105Asn variant exhibited increased K m s for all substrates. In contrast, the Trp105Leu and -Val substituted enzymes demonstrated notably decreased catalytic efficiencies (k cat /K m ) for all substrates. With respect to clavulanic acid, the K i s and partition ratios were increased for the Trp105Phe, -Asn, and -Val variants. We conclude that interactions between Trp105 of KPC-2 and the b-lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in b-lactam and b-lactamase inhibitor discrimination.

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Section: Inhibitor Discriminationmentioning

confidence: 89%

Section: B-lactamase Purificationmentioning

confidence: 99%

Section: Kineticsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Elucidating the role of Trp105 in the KPC‐2 β‐lactamase

et al. 2010

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“…Knowing which variant is present might open new treatment options in the future, especially under strict antibiotic stewardship. The difference in resistance profiles and their effects on beta-lactam inhibitors were demonstrated directly in clinical samples and transformants with KPC variants and through comparisons of hydrolytic activities (1,27,28,37,45). Robledo et al (36) reported a variation of antimicrobial susceptibility to carbapenems depending on the KPC variant during a 1-year study based on KPC-producing isolates taken from 6 Puerto Rico Medical Center hospitals.…”

Section: Discussionmentioning

confidence: 99%

Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

et al. 2012

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; and Microbiology, NHS Lothian, Edinburgh, United Kingdom c Klebsiella pneumoniae carbapenemases (KPCs) are considered a serious threat to antibiotic therapy, as they confer resistance to carbapenems, which are used to treat extended-spectrum beta-lactamase (ESBL)-producing bacteria. Here, we describe the development and evaluation of a DNA microarray for the detection and genotyping of KPC genes (bla KPC ) within a 5-h period. To test the whole assay procedure (DNA extraction plus a DNA microarray assay) directly from clinical specimens, we compared two commercial DNA extraction kits (the QIAprep Spin miniprep kit [Qiagen] and the urine bacterial DNA isolation kit [Norgen]) for the direct DNA extraction from urine samples (dilution series spiked in human urine). Reliable single nucleotide polymorphism (SNP) typing was demonstrated using 1 ؋ 10 5 CFU/ml urine for Escherichia coli (Qiagen and Norgen) and 80 CFU/ml urine, on average, for K. pneumoniae (Norgen). This study presents, for the first time, the combination of a new KPC microarray with commercial sample preparation for detecting and genotyping microbial pathogens directly from clinical specimens; this paves the way toward tests providing epidemiological and diagnostic data, enabling better antimicrobial stewardship.

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A novel KPC-113 variant conferring carbapenem and ceftazidime-avibactam resistance in a multidrug-resistant Pseudomonas aeruginosa isolate

Yang

et al. 2023

Clinical Microbiology and Infection

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Substrate Selectivity and a Novel Role in Inhibitor Discrimination by Residue 237 in the KPC-2 β-Lactamase

Cited by 48 publications

References 60 publications

Elucidating the role of Trp105 in the KPC‐2 β‐lactamase

Elucidating the role of Trp105 in the KPC‐2 β‐lactamase

Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

A novel KPC-113 variant conferring carbapenem and ceftazidime-avibactam resistance in a multidrug-resistant Pseudomonas aeruginosa isolate

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