Structural Studies of Klebsiella pneumoniae Fosfomycin-Resistance Protein and Its Application for the Development of an Optical Biosensor for Fosfomycin Determination
Abstract
:1. Introduction
2. Results and Discussion
2.1. The Expression, Purification, and Substrate Specificity of FosAPA and FosAKP
2.2. Structural Analysis
2.3. Thermal Stability of FosAKP
2.4. Tethering of FosAKP
2.5. Determination of Fos in Tap Water and Sheep Plasma Samples
3. Materials and Methods
3.1. Materials
3.2. Methods
3.2.1. Molecular Cloning
3.2.2. Expression and Purification FosAPA and FosAKP
3.2.3. Assay of the Enzyme Activity and Protein Concentration Determination
3.2.4. Thermal Stability
3.2.5. Crystallization
3.2.6. Structure Determination
3.2.7. FosAKP Tethering via Covalent Cross-Linking
3.2.8. Kinetic Analysis of the Free and Tethered FosAKP
3.2.9. Biosensor Performance and Recovery Tests
3.2.10. Stability Analysis of Free and Immobilized FosAKP
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Data Processing | FosAKP |
---|---|
Beamline | BioMAX (MAX IV, Lund, Sweden) |
Wavelength (Å) | 0.9919 |
Resolution (Å) | 149.2–1.48 (1.51–1.48) |
Space group | P212121 |
Unit cell a, b, c (Å) | 40.6, 47.0, and 149.2 |
No. of molecules/asymmetric unit | 2 |
No. of unique reflections | 48,410 (2174) |
Completeness (%) | 99.4 (92.2) |
Multiplicity | 9.8 (6.3) |
Mosaicity (°) | 0.13 |
Rmeas | 0.089 (1.452) |
CC1/2 | 0.999 (0.666) |
Mean (I/σ(I)) | 11.8 (1.2) |
Wilson B factor (Å2) | 22.2 |
Refinement | |
Resolution (Å) | 44.85–1.48 |
No. of reflections used | 48,242 |
Rcryst/Rfree | 0.173/0.204 |
RMSD in bonds (Å) | 0.005 |
RMSD in angles (°) | 0.899 |
No. of protein atoms | 2203 |
No. of water molecules | 383 |
Average B-factor (Å2) | 27.7 |
Ramachandran favored/outliers (%) | 99.6/0.0 |
Clashscore | 4.6 |
PDB ID | 8R37 |
Buffer System | Tm (°C) |
---|---|
20 mM NaH2PO4 (pH 8.0) | 65.9 ± 0.3 |
20 mM NaH2PO4 (pH 8.0) and 0.05 mM MnCl2 | 67.3 ± 0.1 |
20 mM NaH2PO4 (pH 8.0) and 100 mM KCl | 64.7 ± 0.3 |
20 mM NaH2PO4 (pH 8.0) and 0.05 mM MnCl2/100 mM KCl | 67.5 ± 0.2 |
20 mM NaH2PO4 (pH 7.0) | 63.0 ± 0.2 |
20 mM HEPES (pH 8.0) | 61.9 ± 0.2 |
20 mM MOPS (pH 8.0) | 61.0 ± 0.2 |
10 mM Tris/75 mM NaCl (pH 7.5) | 59.4 ± 0.1 |
20 mM Tris (pH 7.8) | 58.7 ± 0.2 |
20 mM HEPES (pH 7.0) | 57.6 ± 0.1 |
20 mM L-Arg/20 mM L-Glu (pH 7.5) | 56.8 ± 0.3 |
10 mM acetate (pH 4.2) | 53.7 ± 0.0 |
10 mM acetate (pH 5.2) | 44.3 ± 0.0 |
Run Order | Glutaraldehyde (%v/v) | BSA (mg) | Enzyme (units) |
---|---|---|---|
1 | 0.5 | 16.5 | 13.5 |
2 | 0.5 | 16.5 | 6.8 |
3 | 0.5 | 33.0 | 13.5 |
4 | 0.5 | 33.0 | 6.8 |
5 | 0.7 | 16.5 | 13.5 |
6 | 0.7 | 16.5 | 6.8 |
7 | 0.7 | 33.0 | 13.5 |
8 | 0.7 | 33.0 | 6.8 |
9 | 1.0 | 16.5 | 13.5 |
10 | 1.0 | 16.5 | 6.8 |
11 | 1.0 | 33.0 | 13.5 |
12 | 1.0 | 33.0 | 6.8 |
13 | 0.7 | 22.0 | 6.8 |
14 | 1.0 | 22.0 | 6.8 |
Fos Concentration (mM) | Recovery Tests (%) | Reproducibility of Recovery Tests (%) |
---|---|---|
2 | 122.5 ± 4.8 | 6.7 (n = 3) |
4 | 121.7 ± 4.6 | 6.6 (n = 4) |
10 | 88.7 ± 2.3 | 4.6 (n = 3) |
18 | 93.9 ± 4.3 | 8.0 (n = 4) |
Fos Concentration (mM) | Recovery Tests (%) | Reproducibility of Recovery Tests (%) |
---|---|---|
2 | 146.8 ± 0.6 | 0.7 (n = 3) |
4 | 116.8 ± 5.5 | 8.1(n = 3) |
9 | 112.0 ± 1.6 | 2.1 (n = 3) |
28 | 101.6 ± 5.2 | 7.2 (n = 3) |
37 | 102.0 ± 0.06 | 0.62 (n = 3) |
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Varotsou, C.; Ataya, F.; Papageorgiou, A.C.; Labrou, N.E. Structural Studies of Klebsiella pneumoniae Fosfomycin-Resistance Protein and Its Application for the Development of an Optical Biosensor for Fosfomycin Determination. Int. J. Mol. Sci. 2024, 25, 85. https://doi.org/10.3390/ijms25010085
Varotsou C, Ataya F, Papageorgiou AC, Labrou NE. Structural Studies of Klebsiella pneumoniae Fosfomycin-Resistance Protein and Its Application for the Development of an Optical Biosensor for Fosfomycin Determination. International Journal of Molecular Sciences. 2024; 25(1):85. https://doi.org/10.3390/ijms25010085
Chicago/Turabian StyleVarotsou, Christina, Farid Ataya, Anastassios C. Papageorgiou, and Nikolaos E. Labrou. 2024. "Structural Studies of Klebsiella pneumoniae Fosfomycin-Resistance Protein and Its Application for the Development of an Optical Biosensor for Fosfomycin Determination" International Journal of Molecular Sciences 25, no. 1: 85. https://doi.org/10.3390/ijms25010085