Amino Acid Replacement at Position 228 Induces Fluctuation in the Ω-Loop of KPC-3 and Reduces the Affinity against Oxyimino Cephalosporins: Kinetic and Molecular Dynamics Studies
Abstract
:1. Introduction
2. Results
2.1. KPC-3 Mutants
2.2. Cloning and β-Lactamase Purification
2.3. Steady-State Kinetic Parameters
2.4. Circular Dichroism
2.5. Fluorescence Assay
2.6. MD Simulations
3. Discussion
4. Materials and Methods
4.1. Cloning and Site-Saturation Mutagenesis
4.2. Overexpression and Purification of KPC-3 and D228X Mutants
4.3. Determination of Kinetic Parameters
4.4. Fluorescence Emission Spectroscopy
4.5. Circular Dichroism
4.6. Molecular Dynamics (MD) Simulations
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Substrates (β-Lactams) | KPC-3 | D228S | D228W | D228K | D228M | D228L | D228I | D228G | |
---|---|---|---|---|---|---|---|---|---|
Benzylpenicillin | Km (µM) | 144 ± 22 | 97 ± 8 | 187 ± 1 | 75 ± 5 | 248 ± 14 | 20 ± 2 | 25 ± 2 | 70 ± 6 |
kcat (s−1) | 708 ± 15 | 48 ± 2 | 157 ± 4 | 136 ± 6 | 131 ± 5 | 37 ± 1 | 29 ± 1 | 35 ± 2 | |
kcat/Km (µM−1 s−1) | 4.92 | 0.49 | 0.84 | 1.81 | 0.53 | 1.85 | 1.16 | 0.5 | |
Carbenicillin | Km (µM) | 108 ± 10 | 281 ± 12 | 179 ± 10 | 494 ± 18 | 240 ± 15 | 280 ± 30 | 182 ± 8 | 100 ± 5 |
kcat (s−1) | 18 ± 1 | 17 ± 1 | 27 ± 1 | 36 ± 2 | 25 ± 2 | 14 ± 1 | 21 ± 2 | 70 ± 3 | |
kcat/Km (µM−1 s−1) | 0.17 | 0.06 | 0.15 | 0.07 | 0.10 | 0.05 | 0.12 | 0.7 | |
Cefotaxime | Km (µM) | 57 ± 3 | ≥1000 | 373 ± 21 | 629 ± 50 | 238 ± 15 | 390 ± 28 | 430 ± 30 | 950 ± 48 |
kcat (s−1) | 333 ± 12 | N.D. | 108 ± 5 | 68 ± 2 | 931 ± 21 | 34 ± 2 | 41 ± 2 | 244 ± 11 | |
kcat/Km (µM−1 s−1) | 5.84 | N.D. | 0.29 | 0.11 | 3.91 | 0.09 | 0.09 | 0.21 | |
Cefazolin | Km (µM) | 189 ± 25 | 150 ± 8 | 119 ± 11 | 126 ± 14 | 141 ± 18 | 76 ± 6 | 121 ± 15 | 205 ± 17 |
kcat (s−1) | 351 ± 9 | 11 ± 0.4 | 413 ± 3 | 644 ± 8 | 519 ± 14 | 64 ± 4 | 93 ± 4 | 271 ± 8 | |
kcat/Km (µM−1 s−1) | 1.86 | 0.07 | 3.47 | 5.11 | 3.68 | 0.84 | 0.77 | 1.32 | |
Ceftazidime | Km (µM) | 100 ± 15 | >1000 | 400 ± 18 | 506 ± 25 | 300 ± 20 | 321 ± 38 | 960 ± 23 | >1000 |
kcat (s−1) | 1.4 ± 0.1 | N.D. | 3.2 ± 0.1 | 1.8 ± 0.1 | 2.2 ± 0.1 | 0.5 ± 0.1 | 3.7 ± 0.5 | N.D. | |
kcat/Km (µM−1 s−1) | 0.01 | N.D. | 0.01 | 0.004 | 0.007 | 0.002 | 0.004 | N.D. | |
Imipenem | Km (µM) | 88 ± 6 | 83 ± 4 | 128 ± 23 | 88 ± 5 | 87 ± 8 | 110 ± 10 | 104 ± 8 | 50 ± 2 |
kcat(s−1) | 41 ± 2 | 34 ± 1 | 115 ± 9 | 116 ± 5 | 118 ± 2 | 19 ± 1.5 | 30 ± 1 | 26 ± 1 | |
kcat/Km (µM−1 s−1) | 0.46 | 0.41 | 0.89 | 1.32 | 1.36 | 0.17 | 0.29 | 0.52 | |
Meropenem | Km (µM) | 68 ± 3 | 19 ± 2 | 46 ± 1 | 99 ± 5 | 48 ± 3 | 34 ± 1 | 62 ± 2 | 36 ± 1 |
kcat (s−1) | 51 ± 2 | 3 ± 0.1 | 7 ± 0.3 | 8 ± 0.5 | 5 ± 0.3 | 4 ± 0.2 | 5 ± 0.1 | 11 ± 1 | |
kcat/Km(µM−1 s−1) | 0.75 | 0.16 | 0.15 | 0.08 | 0.10 | 0.12 | 0.08 | 0.31 | |
Ertapenem | Km (µM) | 30 ± 1 | 63 ± 4 | 40 ± 3 | 26 ± 1 | 30 ± 2 | 21 ± 1 | 29 ± 2 | 18 ± 1 |
kcat (s−1) | 5 ± 0.5 | 23 ± 2 | 25 ± 1 | 10 ± 1 | 82 ± 1 | 7 ± 0.4 | 11 ± 0.5 | 7 ± 0.2 | |
kcat/Km (µM−1 s−1) | 0.17 | 0.37 | 0.62 | 0.38 | 2.73 | 0.33 | 0.38 | 0.39 |
Enzymes | Alpha | Beta | Turn | Unordered | Total | NRMSD (a) |
---|---|---|---|---|---|---|
KPC-3 | ||||||
CDSSTR | 39 | 17 | 18 | 26 | 100 | 0.055 |
CONTIN | 36 | 16 | 19 | 29 | 100 | 0.036 |
SELCON | 37 | 15 | 19 | 30 | 101 | 0.103 |
AVERAGE | 37 ± 1.5 | 16 ± 1 | 18.7 ± 0.6 | 28 ± 2 | 100 ± 5 | |
D228L | ||||||
CDSSTR | 33 | 21 | 19 | 27 | 100 | 0.061 |
CONTIN | 29 | 20 | 20 | 30 | 99 | 0.049 |
SELCON | 32 | 17 | 20 | 31 | 100 | 0.130 |
AVERAGE | 31 ± 2 | 19 ± 2 | 19.7 ± 0.6 | 29 ± 2 | 100 ± 7 | |
D228K | ||||||
CDSSTR | 34 | 18 | 19 | 28 | 99 | 0.059 |
CONTIN | 31 | 17 | 20 | 31 | 99 | 0.043 |
SELCON | 34 | 17 | 20 | 30 | 101 | 0.120 |
AVERAGE | 33 ± 2 | 17.3 ± 0.6 | 19.7 ± 0.6 | 30 ± 1.5 | 100 ± 5 | |
D228M | ||||||
CDSSTR | 37 | 18 | 18 | 27 | 100 | 0.055 |
CONTIN | 34 | 18 | 19 | 30 | 101 | 0.046 |
SELCON | 35 | 16 | 20 | 30 | 101 | 0.123 |
AVERAGE | 35 ± 1.5 | 17.3 ± 1 | 19 ± 1 | 29 ± 2 | 100 ± 5 | |
GLOBAL AVERAGE | 34 ± 3 | 18 ± 2 | 19 ± 1 | 29 ± 2 | 100 ± 8 |
Specie | Uncomplexed Species | Complexed Species |
---|---|---|
KPC-3 | 29.1 nm | 34.0 nm |
D228S | 32.2 nm | 34.0 nm |
D228M | 36.2 nm | 32.0 nm |
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Piccirilli, A.; Brisdelli, F.; Docquier, J.D.; Aschi, M.; Cherubini, S.; De Luca, F.; Matagne, A.; Amicosante, G.; Perilli, M. Amino Acid Replacement at Position 228 Induces Fluctuation in the Ω-Loop of KPC-3 and Reduces the Affinity against Oxyimino Cephalosporins: Kinetic and Molecular Dynamics Studies. Catalysts 2020, 10, 1474. https://doi.org/10.3390/catal10121474
Piccirilli A, Brisdelli F, Docquier JD, Aschi M, Cherubini S, De Luca F, Matagne A, Amicosante G, Perilli M. Amino Acid Replacement at Position 228 Induces Fluctuation in the Ω-Loop of KPC-3 and Reduces the Affinity against Oxyimino Cephalosporins: Kinetic and Molecular Dynamics Studies. Catalysts. 2020; 10(12):1474. https://doi.org/10.3390/catal10121474
Chicago/Turabian StylePiccirilli, Alessandra, Fabrizia Brisdelli, Jean Denis Docquier, Massimiliano Aschi, Sabrina Cherubini, Filomena De Luca, André Matagne, Gianfranco Amicosante, and Mariagrazia Perilli. 2020. "Amino Acid Replacement at Position 228 Induces Fluctuation in the Ω-Loop of KPC-3 and Reduces the Affinity against Oxyimino Cephalosporins: Kinetic and Molecular Dynamics Studies" Catalysts 10, no. 12: 1474. https://doi.org/10.3390/catal10121474
APA StylePiccirilli, A., Brisdelli, F., Docquier, J. D., Aschi, M., Cherubini, S., De Luca, F., Matagne, A., Amicosante, G., & Perilli, M. (2020). Amino Acid Replacement at Position 228 Induces Fluctuation in the Ω-Loop of KPC-3 and Reduces the Affinity against Oxyimino Cephalosporins: Kinetic and Molecular Dynamics Studies. Catalysts, 10(12), 1474. https://doi.org/10.3390/catal10121474