Interactions of hydrolyzed β-lactams with the L1 metallo-β-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products

Hinchliffe, P.; Calvopiña, Karina; Rabe, Patrick; Mojica, María F; Schofield, ChristopherJ.; Dmitrienko, GaryI.; Bonomo, RobertA.; Vila, Alejandro J.; Spencer, James

doi:10.1016/j.jbc.2023.104606

Cited by 3 publications

(7 citation statements)

References 87 publications

(145 reference statements)

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“…Thus, 1D 1 H NMR differentiates drug binding from drug hydrolysis in the reactions performed with PBP2 or KPC-2 alone, respectively (Figure 3c). 32 Incubation of mecillinam with both PBP2 and KPC-2 in a 1:1 protein-to-drug ratio for both enzymes resulted in the disappearance of 1 H signals corresponding to H 1 , Me 5 , and Me 6 of mecillinam. Thus, PBP2 outcompeted KPC-2, thereby preventing mecillinam hydrolysis.…”

Section: ■ Results and Discussionmentioning

confidence: 98%

“…NMR resonance assignments showed that the H 1 and methyl Me 5 and Me 6 protons of mecillinam were the most affected by β-lactam ring opening (Figure 3b and Figure S11). 32 To evaluate the activity of KPC-2 in the periplasm, mecillinam was mixed with periplasmic extracts from cultures of E. coli strains harboring the vector pTRC99K (negative control) or its derivative encoding KPC-2. Recorded 1D 1 H NMR experiments revealed distinct spectral signatures for the two strains, consistent with hydrolysis of the β-lactam ring by KPC-2 (Figure 3b).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…NMR resonance assignments showed that the H 1 and methyl Me 5 and Me 6 protons of mecillinam were the most affected by β-lactam ring opening (Figure b and Figure S11). …”

Section: Resultsmentioning

confidence: 99%

“…(a) Chemical structures of mecillinam and its hydrolyzed form. Protons, for which NMR resonances were followed during the hydrolysis reaction, were marked in red for mecillinam and in brown for its hydrolyzed form . (b) 1D 1 H T 1ρ -filtered spectra recorded with a spinlock amplitude of 1.7 kHz and a duration of 50 ms at pH 8.0 and 25 °C for the incubation of mecillinam with periplasmic extracts from E. coli strains harboring the vector pTRC99K (no KPC-2 production) or the recombinant plasmid pTRC99KΩ kpc-2 (KPC-2 production).…”

Section: Resultsmentioning

confidence: 99%

“…This indicates that mecillinam was covalently bound to PBP2, causing NMR signals to broaden and disappear from the spectrum. Thus, 1D 1 H NMR differentiates drug binding from drug hydrolysis in the reactions performed with PBP2 or KPC-2 alone, respectively (Figure c) . Incubation of mecillinam with both PBP2 and KPC-2 in a 1:1 protein-to-drug ratio for both enzymes resulted in the disappearance of 1 H signals corresponding to H 1 , Me 5 , and Me 6 of mecillinam.…”

Section: Resultsmentioning

confidence: 99%

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Monitoring Drug–Protein Interactions in the Bacterial Periplasm by Solution Nuclear Magnetic Resonance Spectroscopy

Razew,

Herail,

Miyachiro

et al. 2024

J. Am. Chem. Soc.

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The rapid spread of antimicrobial resistance across bacterial pathogens poses a serious risk to the efficacy and sustainability of available treatments. This puts pressure on research concerning the development of new drugs. Here, we present an in-cell NMR-based research strategy to monitor the activity of the enzymes located in the periplasmic space delineated by the inner and outer membranes of Gram-negative bacteria. We demonstrate its unprecedented analytical power in monitoring in situ and in real time (i) the hydrolysis of β-lactams by β-lactamases, (ii) the interaction of drugs belonging to the β-lactam family with their essential targets, and (iii) the binding of inhibitors to these enzymes. We show that in-cell NMR provides a powerful analytical tool for investigating new drugs targeting the molecular components of the bacterial periplasm.

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Section: ■ Results and Discussionmentioning

confidence: 98%

Section: ■ Results and Discussionmentioning

confidence: 99%

“…NMR resonance assignments showed that the H 1 and methyl Me 5 and Me 6 protons of mecillinam were the most affected by β-lactam ring opening (Figure b and Figure S11). …”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 3 more Smart Citations

Monitoring Drug–Protein Interactions in the Bacterial Periplasm by Solution Nuclear Magnetic Resonance Spectroscopy

Razew,

Herail,

Miyachiro

et al. 2024

J. Am. Chem. Soc.

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SERS study of classical and newly β-lactams-metal complexation based on in situ laser-induced coral reefs-like silver photomicroclusters: In vitro study of antibacterial activity

El-Zahry

Nasr

Al-Shaalan

et al. 2023

Journal of Pharmaceutical and Biomedical Analysis

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A novel pathogenic species of genus Stenotrophomonas: Stenotrophomonas pigmentata sp. nov

Li,

Yu,

Fan

et al. 2024

Front. Cell. Infect. Microbiol.

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IntroductionStenotrophomonas is a prominent genus owing to its dual nature. Species of this genus have many applications in industry and agriculture as plant growth-promoting rhizobacteria and microbial biological control agents, whereas species such as Stenotrophomonas maltophilia are considered one of the leading gram-negative multi-drug-resistant bacterial pathogens because of their high contribution to the increase in crude mortality and significant clinical challenge. Pathogenic Stenotrophomonas species and most clinical isolates belong to the Stenotrophomonas maltophilia complex (SMc). However, a strain highly homologous to S. terrae was isolated from a patient with pulmonary tuberculosis (TB), which aroused our interest, as S. terrae belongs to a relatively distant clade from SMc and there have been no human association reports.MethodsThe pathogenicity, immunological and biochemical characteristics of 610A2T were systematically evaluated.Results610A2T is a new species of genus Stenotrophomonas, which is named as Stenotrophomonas pigmentata sp. nov. for its obvious brown water-soluble pigment. 610A2T is pathogenic and caused significant weight loss, pulmonary congestion, and blood transmission in mice because it has multiple virulence factors, haemolysis, and strong biofilm formation abilities. In addition, the cytokine response induced by this strain was similar to that observed in patients with TB, and the strain was resistant to half of the anti-TB drugs.ConclusionsThe pathogenicity of 610A2T may not be weaker than that of S. maltophilia. Its isolation extended the opportunistic pathogenic species to all 3 major clades of the genus Stenotrophomonas, indicating that the clinical importance of species of Stenotrophomonas other than S. maltophilia and potential risks to biological safety associated with the use of Stenotrophomonas require more attention.

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Interactions of hydrolyzed β-lactams with the L1 metallo-β-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products

Cited by 3 publications

References 87 publications

Monitoring Drug–Protein Interactions in the Bacterial Periplasm by Solution Nuclear Magnetic Resonance Spectroscopy

Monitoring Drug–Protein Interactions in the Bacterial Periplasm by Solution Nuclear Magnetic Resonance Spectroscopy

SERS study of classical and newly β-lactams-metal complexation based on in situ laser-induced coral reefs-like silver photomicroclusters: In vitro study of antibacterial activity

A novel pathogenic species of genus Stenotrophomonas: Stenotrophomonas pigmentata sp. nov

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