Exploring the Role of L10 Loop in New Delhi Metallo-β-lactamase (NDM-1): Kinetic and Dynamic Studies
Abstract
:1. Introduction
2. Results
2.1. Determination of kcat and Km
2.1.1. Imipenem
2.1.2. Meropenem
2.1.3. Benzylpenicillin
2.1.4. Carbenicillin
2.1.5. Cefazolin
2.1.6. Cefoxitin
2.1.7. Cefotaxime and Ceftazidime
2.1.8. Cefepime
2.2. Effect of pH on Km and kcat
2.3. Thermofluor Stability of NDM-1 and Its Mutants
2.4. Fluorescence Spectra
2.5. Molecular Dynamics Simulations
2.6. Molecular Dynamics Simulations for NDM-1 and L218T in Complex with Meropenem
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Site-Directed Mutagenesis
5.2. Cloning of blaL218T, blaL221T, blaL269H and blaL221T/Y229W
5.3. Production and Purification of L218T, L221T, L269H and L221T-Y229W Enzymes
5.4. Determination of Kinetic Parameters
5.5. Fluorescence Assays
5.6. Thermofluor Assay
5.7. Molecular Dynamics Simulations
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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Substrates | Variant | Km (μM) | kcat (s−1) | kcat/Km (M−1 s−1) |
---|---|---|---|---|
Imipenem | NDM-1 * | 35 ± 1 | 64 ± 3 | 1.8 × 106 |
L218T | 13 ± 1 | 2 ± 0.1 | 1.5 × 105 | |
L221T | 59 ± 4 | 8 ± 1 | 1.3 × 105 | |
L269H | 149 ± 9 | 40 ± 3 | 2.7 × 105 | |
L221T/Y229W | 129 ± 6 | 29 ± 1 | 2.2 × 105 | |
Y229W * | 81 ± 3 | 38 ± 1 | 4.7 × 105 | |
Meropenem | NDM-1 * | 80 ± 2 | 75 ± 2 | 9.4 × 105 |
L218T | 22 ± 2 | 1 ± 0.1 | 4.5 × 104 | |
L221T | 20 ± 1 | 9 ± 1 | 4.5 × 105 | |
L269H | 117 ± 7 | 109 ± 8 | 9.3 × 105 | |
L221T/Y229W | 174 ± 6 | 135 ± 3 | 7.7 × 105 | |
Y229W * | 259 ± 18 | 820 ± 5 | 3.2 × 106 | |
Benzylpenicillin | NDM-1 * | 250 ± 10 | 105 ± 5 | 4.2 × 105 |
L218T | 832 ± 20 | 294 ± 2 | 3.5 × 105 | |
L221T | 937 ± 25 | 78 ± 5 | 8.3 × 104 | |
L269H | 541 ± 12 | 549 ± 5 | 1.0 × 106 | |
L221T/Y229W | >2000 | >1000 | 0.29 | |
Y229W * | 841 ± 35 | 552 ± 3 | 6.6 × 105 | |
Carbenicillin | NDM-1 * | 285 ± 5 | 108 ± 4 | 3.8 × 105 |
L218T | 265 ± 10 | 59 ± 3 | 2.2 × 105 | |
L221T | 398 ± 15 | 48 ± 3 | 1.2 × 105 | |
L269H | 339 ± 9 | 347 ± 5 | 1.0 × 106 | |
L221T/Y229W | >2000 | >1000 | 0.29 | |
Y229W * | 1176 ± 25 | 866 ± 6 | 7.3 × 105 | |
Cefazolin | NDM-1 * | 20 ± 1 | 42 ± 1 | 2.1 × 106 |
L218T | 74 ± 5 | 9 ± 1 | 1.2 × 105 | |
L221T | 25 ± 3 | 8 ± 1 | 3.2 × 105 | |
L269H | 41 ± 2 | 83 ± 2 | 2.0 × 106 | |
L221T/Y229W | 9 ± 1 | 14 ± 1 | 1.5 × 106 | |
Y229W* | 48 ± 2 | 191 ± 4 | 4.0 × 106 | |
Cefoxitin | NDM-1 * | 26 ± 1 | 23 ± 1 | 8.8 × 105 |
L218T | 31 ± 3 | 3 ± 0.2 | 9.6 × 104 | |
L221T | 31 ± 3 | 1.5 ± 0.1 | 4.8 × 104 | |
L269H | 100 ± 5 | 11 ± 1 | 1.1 × 105 | |
L221T/Y229W | >5000 | >11 | 3.1 × 103 | |
Y229W * | 2100 ± 25 | 4 ± 0.2 | 2.0 × 103 | |
Cefotaxime | NDM-1 * | 14 ± 1 | 20 ± 1 | 1.4 × 106 |
L218T | 24 ± 2 | 30 ± 2 | 1.2 × 106 | |
L221T | 10 ± 1 | 2 ± 0.1 | 2.0 × 10 | |
L269H | 37 ± 2 | 37 ± 1 | 1.0 × 106 | |
L221T/Y229W | 10 ± 1 | 14 ± 1 | 1.4 × 106 | |
Y229W * | 52 ± 4 | 107 ± 3 | 2.1 × 106 | |
Ceftazidime | NDM-1 * | 50 ± 4 | 18 ± 0.5 | 3.7 × 105 |
L218T | 19 ± 1 | 45 ± 3 | 2.4 × 106 | |
L221T | 32 ± 1 | 19 ± 1 | 5.9 × 105 | |
L269H | 81 ± 4 | 10 ± 1 | 1.2 × 105 | |
L221T/Y229W | 222 ± 10 | 7 ± 1 | 3.0 × 104 | |
Y229W * | 250 ± 15 | 10 ± 1 | 4.0 × 104 | |
Cefepime | NDM-1 * | 35 ± 5 | 13 ± 1 | 3.7 × 105 |
L218T | 39 ± 2 | 3 ± 0.1 | 7.7 × 104 | |
L221T | 40 ± 3 | 2 ± 0.2 | 5.0 × 104 | |
L269H | 64 ± 3 | 11 ± 1 | 1.7 × 105 | |
L221T/Y229W | 152 ± 8 | 3 ± 0.5 | 2.0 × 104 | |
Y229W * | 117 ± 8 | 2.5 ± 0.1 | 2.0 × 104 |
Meropenem | HEPES 20 mM 20 μM ZnCl2 pH 7.00 | BIS TRIS 20 mM 20 μM ZnCl2 pH 6.5 | MES 20 mM 20 μM ZnCl2 pH 6.0 | ||||||
---|---|---|---|---|---|---|---|---|---|
Km (μM) | kcat (s−1) | kcat/Km (μM−1 s−1) | Km (μM) | kcat (s−1) | kcat/Km (μM−1 s−1) | Km (μM) | kcat (s−1) | kcat/Km (μM−1 s−1) | |
NDM-1 | 80 ± 2 | 75 | 0.94 | 46 ± 2 | 18 | 0.39 | 98 ± 3 | 40 | 0.41 |
L218T | 22 ± 2 | 1 | 0.04 | 166 ± 7 | 11 | 0.06 | 189 ± 8 | 15 | 0.08 |
L221T | 20 ± 1 | 9 | 0.45 | 30 ± 2 | 5 | 0.16 | 28 ± 2 | 4 | 0.14 |
L269H | 117 ± 7 | 109 | 0.93 | 42 ± 3 | 96 | 2.28 | 182 ± 7 | 167 | 0.92 |
L221T/Y229W | 174 ± 6 | 135 | 0.77 | 298 ± 18 | 67 | 0.22 | 101 ± 4 | 36 | 0.36 |
Y229W | 259 ± 18 | 820 | 3.17 | 72 ± 4 | 67 | 0.93 | 104 ± 5 | 60 | 0.57 |
Enzymes | TmB (°C) | ΔTmB (°C) | TmD (°C) | ΔTmD (°C) |
---|---|---|---|---|
NDM-1 | 50.66 | − | 53.73 | − |
L218T | 50.38 | −0.28 | 52.41 | 1.32 |
L221T | 49.06 | −1.60 | 46.55 | −7.18 |
L269H | 42.87 | −7.79 | 44.51 | −9.22 |
Y229W | 46.62 | −4.04 | 47.86 | −5.87 |
L221T/Y229W | 45.02 | −5.64 | 46.36 | −7.37 |
NDM-1 | L218T | L221T | L269H | L221T/Y229W | |
---|---|---|---|---|---|
Zn1-Zn2 | 3.26 ± 0.05 | 3.28 ± 0.05 | 3.27 ± 0.05 | 3.18 ± 0.10 | 3.27 ± 0.06 |
Zn1-OH- | 1.70 ± 0.03 | 1.71 ± 0.03 | 1.70 ± 0.03 | 1.68 ± 0.03 | 1.71 ± 0.03 |
Zn2-OH- | 1.66 ± 0.02 | 1.68 ± 0.03 | 1.68 ± 0.03 | 1.69 ± 0.03 | 1.66 ± 0.02 |
Zn1-H120 | 4.30 ± 0.49 | 4.13 ± 0.28 | 4.29 ± 0.29 | 1.84 ± 0.04 | 4.18 ± 0.26 |
Zn1-H122 | 4.98 ± 0.84 | 4.40 ± 0.46 | 4.59 ± 0.47 | 4.37 ± 0.95 | 4.64 ± 0.52 |
Zn1-H189 | 1.96 ± 0.07 | 1.93 ± 0.06 | 1.96 ± 0.07 | 4.27 ± 0.66 | 1.94 ± 0.07 |
Zn2-D124 | 3.27 ± 0.51 | 1.89 ± 0.10 | 2.18 ± 0.63 | 2.90 ± 0.99 | 1.83 ± 0.30 |
Zn2-C208 | 1.95 ± 0.03 | 2.00 ± 0.05 | 1.99 ± 0.05 | 1.98 ± 0.05 | 1.95 ± 0.04 |
Zn2-H250 | 4.26 ± 0.35 | 4.28 ± 0.31 | 4.32 ± 0.32 | 5.19 ± 2.92 | 4.40 ± 0.30 |
NDM-1/Meropenem Complex | L218T/Meropenem Complex | |
---|---|---|
Zn1-Zn2 | 4.54 ± 0.15 | 5.87 ± 0.42 |
Zn1-H120 | 3.63 ± 0.56 | 4.04 ± 0.78 |
Zn1-H122 | 1.95 ± 0.06 | 4.34 ± 0.98 |
Zn1-H189 | 2.05 ± 0.10 | 1.92 ± 0.07 |
Zn2-D124 | 1.73 ± 0.04 | 1.84 ± 0.33 |
Zn2-C208 | 1.93 ± 0.03 | 1.96 ± 0.04 |
Zn2-H250 | 1.97 ± 0.07 | 2.36 ± 0.31 |
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Piccirilli, A.; Criscuolo, E.; Brisdelli, F.; Mercuri, P.S.; Cherubini, S.; De Sciscio, M.L.; Maccarrone, M.; Galleni, M.; Amicosante, G.; Perilli, M. Exploring the Role of L10 Loop in New Delhi Metallo-β-lactamase (NDM-1): Kinetic and Dynamic Studies. Molecules 2021, 26, 5489. https://doi.org/10.3390/molecules26185489
Piccirilli A, Criscuolo E, Brisdelli F, Mercuri PS, Cherubini S, De Sciscio ML, Maccarrone M, Galleni M, Amicosante G, Perilli M. Exploring the Role of L10 Loop in New Delhi Metallo-β-lactamase (NDM-1): Kinetic and Dynamic Studies. Molecules. 2021; 26(18):5489. https://doi.org/10.3390/molecules26185489
Chicago/Turabian StylePiccirilli, Alessandra, Emanuele Criscuolo, Fabrizia Brisdelli, Paola Sandra Mercuri, Sabrina Cherubini, Maria Laura De Sciscio, Mauro Maccarrone, Moreno Galleni, Gianfranco Amicosante, and Mariagrazia Perilli. 2021. "Exploring the Role of L10 Loop in New Delhi Metallo-β-lactamase (NDM-1): Kinetic and Dynamic Studies" Molecules 26, no. 18: 5489. https://doi.org/10.3390/molecules26185489