Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases
Abstract
:1. Introduction
2. Results
2.1. EnpACD Structure Determination
2.2. Overall Structure of EnpACD
2.3. The Architecture of Active Site and Binding Groove
2.4. Structural Comparison with Other Members of M23 Family
2.5. Specificity and Bacteriolytic Activity of EnpACD
2.6. Substrate Binding Model to EnpACD Reveals Possible Interactions
3. Discussion
3.1. EnpACD Displays a Potent Antibacterial Activity
3.2. General Architecture of the Binding Groove of M23 Reflects Enzyme Specificity
3.3. Arginine 21 Plays a Role in EnpA Activity
4. Materials and Methods
4.1. Gene Cloning
4.2. Mutagenesis
4.3. Protein Purification
4.4. EnpACD H109A Crystallization
4.5. Data Collection, Processing
4.6. Structure Determination and Refinement
4.7. Modelling of the EnpACD Substrate Complex
4.8. Enzymatic Activity Assays
4.9. FTIR
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Małecki, P.H.; Mitkowski, P.; Jagielska, E.; Trochimiak, K.; Mesnage, S.; Sabała, I. Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases. Int. J. Mol. Sci. 2021, 22, 7136. https://doi.org/10.3390/ijms22137136
Małecki PH, Mitkowski P, Jagielska E, Trochimiak K, Mesnage S, Sabała I. Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases. International Journal of Molecular Sciences. 2021; 22(13):7136. https://doi.org/10.3390/ijms22137136
Chicago/Turabian StyleMałecki, Piotr Henryk, Paweł Mitkowski, Elżbieta Jagielska, Karolina Trochimiak, Stéphane Mesnage, and Izabela Sabała. 2021. "Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases" International Journal of Molecular Sciences 22, no. 13: 7136. https://doi.org/10.3390/ijms22137136