What question did this study set out to answer?

To elucidate how amino acid substitutions in the putative omega-like loop region near the active site influence the hydrolytic activity and stability of the NDM-5 beta-lactamase.

January 12, 2017FEMS Microbiology Letters

E152A substitution drastically affects NDM-5 activity

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Authors

GKGaurav KumarSRM Institute of Science and Technology BIBagre IssaUniversité Félix Houphouët-Boigny DKDebasish KarM S Ramaiah University of Applied Sciences

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Implication

In vitro mutational analysis reveals that the E152A substitution drastically impairs beta-lactam hydrolysis in NDM-5, suggesting the loop residue is vital for carbapenemase function.

Key Points

To elucidate how amino acid substitutions in the putative omega-like loop region near the active site influence the hydrolytic activity and stability of the NDM-5 beta-lactamase.
Engineered three active-site-adjacent point mutations (E152A, S191A, and D223A) in NDM-5 and expressed each variant in a bacterial host to evaluate changes in antimicrobial resistance.
Purified wild-type NDM-5 and the E152A mutant to analyze enzymatic hydrolysis kinetics (Km, kcat/Km), overall protein folding, and thermal stability profiles across multiple beta-lactam classes.
The E152A substitution markedly reduced host resistance against beta-lactam antibiotics compared to wild-type NDM-5, whereas S191A and D223A did not elicit comparable reductions.
Enzyme kinetic analyses demonstrated suppressed hydrolytic activity, lower substrate affinity (increased Km), and diminished catalytic efficiency (kcat/Km) against penicillins and carbapenems for NDM-5_E152A.
Circular dichroism and biophysical analyses revealed significant shifts in thermal stability for the E152A mutant relative to wild-type NDM-5, despite maintaining the overall native folding pattern.

Cite This Study

Kumar et al. (2017) studied this question.

synapsesocial.com/papers/6a857353b61bc202bc1e59c8 https://doi.org/10.1093/femsle/fnx008

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Implication

In vitro mutational analysis reveals that the E152A substitution drastically impairs beta-lactam hydrolysis in NDM-5, suggesting the loop residue is vital for carbapenemase function.

E152A substitution drastically affects NDM-5 activity

Discussion

Member takes

Implication

Key Points

Cite This Study

E152A substitution drastically affects NDM-5 activity

Discussion

Member takes

Implication

Key Points

Cite This Study

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