Structural Analysis of Plasmodium falciparum Hexokinase Provides Novel Information about Catalysis Due to a Plasmodium-Specific Insertion
Abstract
:1. Introduction
2. Results
2.1. Overall Structure of P. falciparum Hexokinase
2.2. Glucose and Citrate Were Bound to the Active Site
2.3. A Plasmodium-Specific Insertion (P-Insert) Lined the Active Site
2.4. Does Catalysis Affect the Conformation of the C-Loop and the P-Insert?
2.5. Structural Comparisons Suggest a Plasmodium-Specific Mechanism
2.6. Oligomerization of P. falciparum Hexokinase
2.7. Oxidative Posttranslational Modifications Inhibit the Enzymatic Activity of PfHK
3. Discussion
4. Materials and Methods
4.1. Heterologous Expression and Purification of Recombinant PfHK
4.2. Kinetic Characterization of PfHK
4.3. Crystallization
4.4. Data Collection and Refinement
4.5. Gel Filtration Chromatography
4.6. Glutathionylation
4.7. Synthesis of S-Nitrosocysteine, Nitrosation, and Biotin-Switch Assay
4.8. Mass Spectrometry Analysis
4.9. Data Analyses and Statistics
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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PDB Accession Code | 7ZZI (PfHK) |
---|---|
Space group | P3121 |
Unit cell parameters | |
a, b, c (Å) | 125.24, 125.24, 120.93 |
α, β, γ (°) | 90, 90, 120 |
Data collection | |
Beamline | SLS beam line X10SA |
Temperature (K) | 100 |
Resolution range | 49.5–2.8 (2.9–2.8) |
Wilson B-factor Å2 | 95.8 |
Total reflections | 278,108 (26,474) |
Unique reflections | 27,459 (2644) |
Multiplicity | 10.1 (10.0) |
Completeness (%) | 99.7 (97.1) |
Mean I/σ (I) | 13.1 (0.8) |
Wilson B-factor | 95.8 |
R-merge (%) | 0.12 (3.41) |
R-pim (%) | 0.04 (1.12) |
CC1/2 (%) | 0.999 (0.305) |
Molecules per ASU | 2 |
Refinement | |
Reflections used in refinement | 27,456 (2644) |
Reflections used for R-free | 2746 (264) |
R-work | 0.199 (0.428) |
R-free | 0.268 (0.481) |
Protein residues | 930 |
Macromolecules average B-factor Å2 | 89.9 |
Ligands average B-factor Å2 | 82.8 |
Solvent average B-factor Å2 | 82.2 |
RMS deviations | |
Bonds (Å) | 0.01 |
Angles (°) | 1.17 |
Ramachandran favored (%) | 96.11 |
KM (mM) | ||
---|---|---|
Glucose | ATP | |
PfHK, present study | 0.13 ± 0.01 | 0.83 ± 0.08 |
PfHK, Harris et al. 2013 [7] | 0.62 ± 0.06 | 0.66 ± 0.08 |
PfHK, Roth et al. 1987 [12] * | 0.431 ± 0.021 | 3.1 ± 1.4 |
CpHK, Yu et al. 2015 [43] | 0.138 | 0.673 |
TgHK, Saito et al. 2002 [42] | 0.0080 ± 0.0008 | 1.05 ± 0.25 |
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Dillenberger, M.; Werner, A.-D.; Velten, A.-S.; Rahlfs, S.; Becker, K.; Fritz-Wolf, K. Structural Analysis of Plasmodium falciparum Hexokinase Provides Novel Information about Catalysis Due to a Plasmodium-Specific Insertion. Int. J. Mol. Sci. 2023, 24, 12739. https://doi.org/10.3390/ijms241612739
Dillenberger M, Werner A-D, Velten A-S, Rahlfs S, Becker K, Fritz-Wolf K. Structural Analysis of Plasmodium falciparum Hexokinase Provides Novel Information about Catalysis Due to a Plasmodium-Specific Insertion. International Journal of Molecular Sciences. 2023; 24(16):12739. https://doi.org/10.3390/ijms241612739
Chicago/Turabian StyleDillenberger, Melissa, Anke-Dorothee Werner, Ann-Sophie Velten, Stefan Rahlfs, Katja Becker, and Karin Fritz-Wolf. 2023. "Structural Analysis of Plasmodium falciparum Hexokinase Provides Novel Information about Catalysis Due to a Plasmodium-Specific Insertion" International Journal of Molecular Sciences 24, no. 16: 12739. https://doi.org/10.3390/ijms241612739