Inhibition and Biochemical Characterization of Methicillin-Resistant Staphylococcus aureus Shikimate Dehydrogenase: An in Silico and Kinetic Study
Abstract
:1. Introduction
2. Results and Discussion
2.1. Biochemical Characterization of SaSDH
2.2. Kinetic Constants and Reaction Mechanism
Substrate | Km (μM) | Vmax (μmol/min/mg) | Kcat (s−1) | Kcat/Km (M−1x s−1) | Kia | Kia/Km |
---|---|---|---|---|---|---|
Shikimate | 37.46 ± 3.7 | 233.6 ± 1.5 | 135.81 | 3.62 × 106 | 21.224 | 1.76 |
NADP+ | 42.55 ± 9.9 | 251.41 ± 7.2 | 146.16 | 3.43 × 106 | 21.015 | 2.02 |
2.3. SaSDH Inhibition
2.3.1. Homology Modeling and Virtual SCREENING
Compound Structure | Molecular Weight a | H-bond Donor a | H-bond Acceptor a | LogP a | Drug Likeness a, b | Binding energy kcal/mol | % Inhibition 200 µM |
---|---|---|---|---|---|---|---|
894 | 150.1 | 1 | 3 | 0.97 | 0.13 | −11.93 | 99 |
766 | 206.2 | 1 | 3 | 2.43 | −0.76 | −12.55 | 87 |
238 | 210.3 | 1 | 4 | 3.32 | −099 | −12.38 | 87 |
626 | 270.2 | 1 | 2 | 2.83 | −0.40 | −12.85 | 49 |
463 | 195.3 | 1 | 3 | 1.05 | 0.24 | −11.89 | 45 |
62 | 208.2 | 1 | 4 | 2.69 | −0.70 | −11.98 | 33 |
291 | 164.2 | 1 | 2 | 1.66 | −0.80 | −12.27 | 31 |
692 | 273.2 | 1 | 4 | 3.87 | −0.99 | −11.90 | 31 |
306 | 221.2 | 1 | 4 | 1.23 | 0.20 | −11.90 | 31 |
637 | 192.2 | 1 | 3 | 2.83 | −0.79 | −12.25 | 29 |
2.3.2. Kinetic Study of the SaSDH-Inhibitor Complex
Substrate | Inhibitor | Kic (μM) | Kiu (μM) | i0.5 at 400 μM of substrate | Pattern |
---|---|---|---|---|---|
Shikimate | 238 | 9.53 | NA | 122.94 | Competitive |
NADP+ | 238 | ND | 48.3 | 107.52 | Uncompetitive |
Shikimate | 766 | 19.76 | NA | 343.43 | Competitive |
NADP+ | 766 | ND | 11.35 | 564.2 | Uncompetitive |
Shikimate | 894 | 58.3 | 1470 | 142.9 | Mixed-competitive |
NADP+ | 894 | 900.9 | 5.2 | 614 | Mixed-uncompetitive |
2.3.3 Flexible Docking of the SaSDH-Inhibitor Complex
3. Experimental
3.1. SaSDH Gene Cloning
3.2. Enzyme Purification
3.3. Molecular Weight Determination
3.4. Enzyme Activity
3.5. Reaction Mechanism and Kinetic Parameters
3.6. Inhibition Assays
3.7. Biochemical Determinations
3.8. Homology Modeling
3.9. Virtual Screening
3.10. Induced Fit Docking (IFD)
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Avitia-Domínguez, C.; Sierra-Campos, E.; Salas-Pacheco, J.M.; Nájera, H.; Rojo-Domínguez, A.; Cisneros-Martínez, J.; Téllez-Valencia, A. Inhibition and Biochemical Characterization of Methicillin-Resistant Staphylococcus aureus Shikimate Dehydrogenase: An in Silico and Kinetic Study. Molecules 2014, 19, 4491-4509. https://doi.org/10.3390/molecules19044491
Avitia-Domínguez C, Sierra-Campos E, Salas-Pacheco JM, Nájera H, Rojo-Domínguez A, Cisneros-Martínez J, Téllez-Valencia A. Inhibition and Biochemical Characterization of Methicillin-Resistant Staphylococcus aureus Shikimate Dehydrogenase: An in Silico and Kinetic Study. Molecules. 2014; 19(4):4491-4509. https://doi.org/10.3390/molecules19044491
Chicago/Turabian StyleAvitia-Domínguez, Claudia, Erick Sierra-Campos, José Manuel Salas-Pacheco, Hugo Nájera, Arturo Rojo-Domínguez, Jorge Cisneros-Martínez, and Alfredo Téllez-Valencia. 2014. "Inhibition and Biochemical Characterization of Methicillin-Resistant Staphylococcus aureus Shikimate Dehydrogenase: An in Silico and Kinetic Study" Molecules 19, no. 4: 4491-4509. https://doi.org/10.3390/molecules19044491