Mechanistic Insights into β-Lactamase-Catalysed Carbapenem Degradation Through Product Characterisation

Lohans, Christopher T.; Freeman, Emily I.; Groesen, Emma van; Tooke, C.L.; Hinchliffe, P.; Spencer, James; Brem, Jürgen; Schofield, Christopher J.

doi:10.1038/s41598-019-49264-0

Cited by 34 publications

(77 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…We monitored product formation using the signal corresponding to the C-9 methyl group, which manifests distinctive resonances for the carbapenem starting material, the hydrolysis products, and the β-lactone products (1.22, 1.18, and 1.55 ppm, respectively) (14,15). It should be noted that, in principle, the hydrolysis and lactone products can be produced in two epimeric 1-pyrroline forms [i.e., (S)-Δ 1 imine and (R)-Δ 1 imine], as well as the tautomeric 2-pyrroline (Δ 2 enamine) form ( Figure S5A).…”

Section: Nmr Turnover Assaysmentioning

confidence: 99%

“…It should be noted that, in principle, the hydrolysis and lactone products can be produced in two epimeric 1-pyrroline forms [i.e., (S)-Δ 1 imine and (R)-Δ 1 imine], as well as the tautomeric 2-pyrroline (Δ 2 enamine) form ( Figure S5A). Recent work has indicated that the Δ 2 enamine is likely the predominant nascent enzymatic product with most class D SBLs, including OXA-48 and OXA-23 (15). In the case of the products derived from 1βmethyl substituted carbapenems, rapid tautomerization yields the (R)-Δ 1 imine product as the kinetic imine product (which is likely, at least predominantly, a non-enzymatic product) with subsequent conversion to the more thermodynamically stable (S)-Δ 1 imine product ( Figure S5B) (15).…”

Section: Nmr Turnover Assaysmentioning

confidence: 99%

“…Recent work has indicated that the Δ 2 enamine is likely the predominant nascent enzymatic product with most class D SBLs, including OXA-48 and OXA-23 (15). In the case of the products derived from 1βmethyl substituted carbapenems, rapid tautomerization yields the (R)-Δ 1 imine product as the kinetic imine product (which is likely, at least predominantly, a non-enzymatic product) with subsequent conversion to the more thermodynamically stable (S)-Δ 1 imine product ( Figure S5B) (15). Under our standard NMR assay conditions with the 1β-methyl substituted carbapenem meropenem, the (S)-Δ 1 imine product was usually the major product form observed for both the hydrolysis and -lactone products, within the time-frames of our assays ( Figure S5).…”

Section: Nmr Turnover Assaysmentioning

confidence: 99%

“…The C-6 hydroxyethyl side chain is in green. Note, there are different possible tautomeric states of the carbapenem derived pyrroline ring, both in the AEC and for the products; in the case of both hydrolysis and lactone forming reactions the major nascent product is likely the enamine tautomer ( Figure S5) (15). B. Crystallographically derived views of the active site of OXA-48 after reaction with imipenem (white) (PDB 5qb4) (18).…”

Section: Nmr Spectroscopy For Nmr Experiments Amentioning

confidence: 99%

See 3 more Smart Citations

Analysis of β-lactone formation by clinically observed carbapenemases informs on a novel antibiotic resistance mechanism

Aertker

Chan

Lohans

et al. 2020

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

An important mechanism of resistance to β-lactam antibiotics is via their β-lactamase catalyzed hydrolysis. Recent work has shown that, in addition to the established hydrolysis products, the reaction of the class D nucleophilic serine β-lactamases (SBLs) with carbapenems also produces β-lactones. We report studies on the factors determining βlactone formation by class D SBLs. We show that variations in hydrophobic residues at the active site of class D SBLs (i.e., Trp105, Val120, and Leu158, using OXA-48 numbering) impact on the relative levels of β-lactones and hydrolysis products formed. Some variants, i.e., the OXA-48 V120L and OXA-23 V128L variants, catalyze increased βlactone formation compared to the wild-type enzymes. The results of kinetic and product studies reveal that variations of residues other than those directly involved in catalysis, including those arising from clinically observed mutations, can alter the reaction outcome of class D SBL catalysis. NMR studies show some Class D SBLs variants catalyze formation of β-lactones from all clinically relevant carbapenems regardless of the presence or not of a 1β-methyl substituent. Analysis of reported crystal structures for carbapenems derived acylenzyme complexes reveals preferred conformations for hydrolysis and β-lactone formation. The observation of increased β-lactone formation by class D SBLs variants, including the clinically observed carbapenemase OXA-48 V120L, supports the proposal that class D SBL-catalyzed rearrangement of β-lactams to β-lactones is important as a resistance mechanism.

show abstract

Section: Nmr Turnover Assaysmentioning

confidence: 99%

Section: Nmr Turnover Assaysmentioning

confidence: 99%

Section: Nmr Turnover Assaysmentioning

confidence: 99%

Section: Nmr Spectroscopy For Nmr Experiments Amentioning

confidence: 99%

See 2 more Smart Citations

Analysis of β-lactone formation by clinically observed carbapenemases informs on a novel antibiotic resistance mechanism

Aertker

Chan

Lohans

et al. 2020

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…According to sequence similarity, the beta-lactamases can be classified into four major classes distinguished by the serine residue at the active site, and comprises class A, C and D, whereas class B are metallo-proteinases, which require zinc for their activity [10, 11]. The class A and D active sites of an enzyme resemble each other, showing similarity in their conserved residues [12].…”

Section: Introductionmentioning

confidence: 99%

Molecular docking, dynamics and free energy analyses of Acinetobacter baumannii OXA class enzymes with carbapenems investigating their hydrolytic mechanisms

Ramachandran

Jeyakanthan

Lopes

2020

Journal of Medical Microbiology

View full text Add to dashboard Cite

Introduction. Acinetobacter baumannii is a critical priority pathogen listed by the World Health Organization due to increasing levels of resistance to carbapenem classes of antibiotics. It causes wound and other nosocomial infections, which can be life-threatening. Hence, there is an urgent need for the development of new classes of antibiotics. Aim. To study the interaction of carabapenems with class D beta-lactamases (oxacillinases) and analyse drug resistance by studying enzyme–substrate complexes using modelling approaches as a means of establishing correlations with the phenotypic data. Methodology. The three-dimensional structures of carbapenems (doripenem, ertapenem, imipenem and meropenem) were obtained from DrugBank and screened against class D beta-lactamases. Further, the study was extended with their variants. The variants’ structure was homology-modelled using the Schrödinger Prime module (Schrödinger LLC, NY, USA). Results. The first discovered intrinsic beta-lactamase of Acinetobacter baumannii , OXA-51, had a binding energy value of −40.984 kcal mol−1, whereas other OXA-51 variants, such as OXA-64, OXA-110 and OXA-111, have values of −60.638, –66.756 and −67.751 kcal mol−1, respectively. The free energy values of OXA-51 variants produced better results than those of other groups. Conclusions. Imipenem and meropenem showed MIC values of 2 and 8 µg ml−1, respectively against OXA-51 in earlier studies, indicating that these are the most effective drugs for treatment of A. baumannii infection. According to our results, OXA-51 is an active enzyme that shows better interactions and is capable of hydrolyzing carbapenems. When correlating the hydrogen-bonding interaction with MIC values, the predicted results are in good agreement and might provide initial insights into performing similar studies related to OXA variants or other antibiotic–enzyme-based studies.

show abstract

Analysis of Metallo-β-lactamases, oprD Mutation, and Multidrug Resistance of β-lactam Antibiotic-Resistant Strains of Pseudomonas aeruginosa Isolated from Southern China

Chen

Lou

et al. 2020

Curr Microbiol

View full text Add to dashboard Cite

The purpose of this study was to analyze the metallo-β-lactamases (MBLs) genotype and oprD mutations of the β-lactam antibiotic-resistant Pseudomonas aeruginosa (PA) strains isolated from southern China. We collected 110 strains of β-lactam antibiotic-resistant PA from 2 hospitals during January 2016–December 2017 from Dongguan, South China. MBLs were detected, amplified, and typed using EDTA disc synergy test, PCR, and Sanger gene sequencing. The mutations and expression levels of oprD were detected using Sanger gene sequencing and qPCR. A total of 16.36% (18/110) β-lactam antibiotic-resistant PA strains produced MBLs, and the main genotypes of MBLs were IMP-25, VIM-2, and SIM-2. Sanger gene sequencing results showed that 107 of the 110 strains harbored mutations in oprD sequence, while 3 strains were negative for oprD amplification (2.73%). Among the 107 strains with positive amplification (97.27%), the rate of intentional mutations (including deletions, insertions, and premature stop codons) was 93.46% (100/107) and that of no disrupted mutation was 6.54% (7/107). qPCR analysis confirmed that the expression level of the OprD protein in the 7 strains of no disrupted mutation was significantly reduced. Among the β-lactam antibiotic-resistant PA strains in southern China, 16.36% were positive for MBLs. The loss rate of oprD was 2.73%, and almost all PA strains showed oprD amplification variation or transcription downregulation. Thus, impaired oprD expression and MBLs production may be some of the mechanisms of β-lactam antibiotic-resistance of PA strains in southern China.

show abstract

Mechanistic Insights into β-Lactamase-Catalysed Carbapenem Degradation Through Product Characterisation

Cited by 34 publications

References 26 publications

Analysis of β-lactone formation by clinically observed carbapenemases informs on a novel antibiotic resistance mechanism

Analysis of β-lactone formation by clinically observed carbapenemases informs on a novel antibiotic resistance mechanism

Molecular docking, dynamics and free energy analyses of Acinetobacter baumannii OXA class enzymes with carbapenems investigating their hydrolytic mechanisms

Analysis of Metallo-β-lactamases, oprD Mutation, and Multidrug Resistance of β-lactam Antibiotic-Resistant Strains of Pseudomonas aeruginosa Isolated from Southern China

Contact Info

Product

Resources

About