Deciphering the Role of V88L Substitution in NDM-24 Metallo-β-Lactamase
Abstract
:1. Introduction
2. Results and Discussion
2.1. Phylogenetic Analysis
2.2. Functional Study
2.3. Characteristics of Enzyme Activity
2.4. Thermal Stability
2.5. Structure Analysis
3. Material and Methods
3.1. Site-Directed Mutagenesis, Cloning and Expression of NDM Variants
3.2. Antimicrobial Susceptibility Tests
3.3. Production and Purification of NDM-1, NDM-5, and NDM-24
3.4. Determination of Kinetic Parameters
3.5. Circular Dichroism and Structure Analysis
3.6. Thermal Stability Testing
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Antibiotic | MIC (μg/mL) | |||
---|---|---|---|---|
E. coli DH5α/pHSG398 | E. coli DH5α/pHSG398-NDM-24 | E. coli DH5α/pHSG398-NDM-1 | E. coli DH5α/pHSG398-NDM-5 | |
Ampicillin | 2 | >256 | >256 | >256 |
Penicillin G | 16 | >256 | >256 | >256 |
Aztreonam | 0.031 | 0.031 | 0.031 | 0.031 |
Cefepime | 0.031 | 2 | 1 | 2 |
Cefotaxime | 0.062 | 32 | 64 | 64 |
Cefoxitin | 2 | 128 | 128 | 128 |
Ceftazidime | 0.125 | 256 | 256 | 256 |
Cefazolin | 2 | 128 | 128 | 256 |
Ertapenem | 0.015 | 1 | 0.25 | 2 |
Imipenem | 0.062 | 2 | 2 | 2 |
Meropenem | 0.031 | 1 | 1 | 2 |
Kinetic Parameters | Enzyme | β-Lactams b | ||||||
---|---|---|---|---|---|---|---|---|
AMP | PEN | TAG | FEP | MEM | IPM | ETP | ||
Km(μM) | NDM-24 | 638.79 ± 23.86 | 331.30 ± 29.43 | 173.85 ± 9.73 | 318.93 ± 10.86 | 266.24 ± 27.03 | 338.20 ± 24.23 | 125.23 ± 19.08 |
NDM-1 | 1249.98 ± 210.94 | 224.57 ± 13.57 | 213.90 ± 11.01 | 173.55 ± 19.46 | 284.24 ± 7.87 | 234.83 ± 7.44 | 105.54 ± 3.09 | |
NDM-5 | 825.00 ± 0.29 | 315.21 ± 46.68 | 76.45 ± 4.76 | 179.64 ± 12.19 | 275.16 ± 36.87 | 292.97 ± 13.76 | 82.18 ± 3.86 | |
kcat (s−1) | NDM-24 | 259.94 ± 23.52 | 179.10 ± 8.17 | 43.13 ± 1.06 | 22.98 ± 0.34 | 151.75 ± 6.69 | 173.16 ± 8.83 | 110.31 ± 7.62 |
NDM-1 | 254.34 ± 28.96 | 79.28 ± 1.96 | 26.73 ± 0.71 | 8.42 ± 0.63 | 75.18 ± 3.44 | 79.81 ± 5.15 | 62.89 ± 1.15 | |
NDM-5 | 346.13 ± 31.30 | 214.13 ± 12.11 | 26.96 ± 0.75 | 13.05 ± 0.24 | 142.48 ± 17.91 | 149.63 ± 2.02 | 83.18 ± 1.67 | |
kcat/Km (μM−1 s−1) | NDM-24 | 0.41 | 0.54 | 0.25 | 0.072 | 0.57 | 0.51 | 0.88 |
NDM-1 | 0.20 | 0.35 | 0.13 | 0.046 | 0.26 | 0.34 | 0.60 | |
NDM-5 | 0.40 | 0.68 | 0.35 | 0.073 | 0.52 | 0.51 | 1.01 | |
kcat/Km (μM−1 s−1) ratio for: | NDM-24/NDM-1 | 2.00 | 1.53 | 1.98 | 1.49 | 2.16 | 1.51 | 1.46 |
NDM-5/NDM-24 | 1.03 | 1.26 | 1.42 | 1.01 | 0.91 | 1.00 | 1.15 | |
NDM-5/NDM-1 | 2.07 | 1.92 | 2.82 | 1.50 | 1.96 | 1.50 | 1.68 |
Program Algorithms a | Structural Elements b | SMP50(9) c | SP37(3) c | SP29(1) c | ||||||
---|---|---|---|---|---|---|---|---|---|---|
NDM-1 | NDM-5 | NDM-24 | NDM-1 | NDM-5 | NDM-24 | NDM-1 | NDM-5 | NDM-24 | ||
SELCON3 | H(r) | 0.070 | 0.078 | 0.092 | 0.062 | 0.074 | 0.092 | 0.059 | 0.079 | 0.087 |
H(d) | 0.085 | 0.088 | 0.089 | 0.081 | 0.088 | 0.089 | 0.078 | 0.087 | 0.086 | |
S(r) | 0.215 | 0.199 | 0.195 | 0.228 | 0.214 | 0.195 | 0.231 | 0.191 | 0.196 | |
S(d) | 0.115 | 0.109 | 0.108 | 0.117 | 0.113 | 0.108 | 0.118 | 0.107 | 0.108 | |
Trn | 0.214 | 0.211 | 0.194 | 0.218 | 0.214 | 0.194 | 0.226 | 0.214 | 0.215 | |
Unrd | 0.284 | 0.287 | 0.261 | 0.282 | 0.279 | 0.261 | 0.287 | 0.292 | 0.285 | |
H(r)+H(d) | 0.155 | 0.166 | 0.181 | 0.143 | 0.162 | 0.181 | 0.137 | 0.166 | 0.173 | |
S(r)+S(d) | 0.33 | 0.308 | 0.303 | 0.345 | 0.327 | 0.303 | 0.349 | 0.298 | 0.304 | |
CONTINLL | H(r) | 0.054 | 0.075 | 0.091 | 0.046 | 0.079 | 0.097 | 0.071 | 0.078 | 0.093 |
H(d) | 0.079 | 0.092 | 0.101 | 0.089 | 0.095 | 0.103 | 0.092 | 0.096 | 0.100 | |
S(r) | 0.217 | 0.208 | 0.187 | 0.202 | 0.205 | 0.182 | 0.197 | 0.197 | 0.183 | |
S(d) | 0.114 | 0.113 | 0.108 | 0.112 | 0.111 | 0.107 | 0.113 | 0.111 | 0.107 | |
Trn | 0.233 | 0.220 | 0.220 | 0.248 | 0.216 | 0.216 | 0.231 | 0.222 | 0.225 | |
Unrd | 0.303 | 0.292 | 0.293 | 0.304 | 0.293 | 0.294 | 0.297 | 0.297 | 0.292 | |
H(r)+H(d) | 0.133 | 0.167 | 0.192 | 0.135 | 0.174 | 0.200 | 0.163 | 0.174 | 0.193 | |
S(r)+S(d) | 0.331 | 0.321 | 0.295 | 0.314 | 0.316 | 0.289 | 0.310 | 0.308 | 0.290 |
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Liu, Z.; Piccirilli, A.; Liu, D.; Li, W.; Wang, Y.; Shen, J. Deciphering the Role of V88L Substitution in NDM-24 Metallo-β-Lactamase. Catalysts 2019, 9, 744. https://doi.org/10.3390/catal9090744
Liu Z, Piccirilli A, Liu D, Li W, Wang Y, Shen J. Deciphering the Role of V88L Substitution in NDM-24 Metallo-β-Lactamase. Catalysts. 2019; 9(9):744. https://doi.org/10.3390/catal9090744
Chicago/Turabian StyleLiu, Zhihai, Alessandra Piccirilli, Dejun Liu, Wan Li, Yang Wang, and Jianzhong Shen. 2019. "Deciphering the Role of V88L Substitution in NDM-24 Metallo-β-Lactamase" Catalysts 9, no. 9: 744. https://doi.org/10.3390/catal9090744