Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase—Insights into the Catalytic Cycle of Bacterial Peroxidases
Abstract
:1. Introduction
2. Results
2.1. Inhibition Studies—Binding and Steady-State Kinetics
2.2. Crystallization of the Active and Azide Inhibited NgBCCP
2.3. Structural Analysis of the Active NgBCCP
2.4. Dimer Interface
2.5. The Active Site
2.6. The Azide-Inhibited NgBCCP
3. Discussion
3.1. NgBCCP Inhibiting Compounds
3.2. NgBCCP Crystal Structure
3.3. The Catalytic Mechanism
4. Materials and Methods
4.1. NgBCCP Production and Purification
4.2. UV-Visible Spectroscopy
4.3. Steady-State Kinetics with Inhibitors
4.4. Crystallization of NgBCCP in the Mixed-Valence State
4.5. Data Collection and Processing
4.6. Structure Solution and Refinement of Preliminary Model
4.7. Protein Structure and Surface Analysis
5. 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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NgBCCP (Active, 1.8 Å) | NgBCCP (Active, 1.4 Å) | NgBCCP (Azide-Inhibited, 2.3 Å) | |
---|---|---|---|
Data Collection and Processing | |||
Source | IμS 3.0 | PXIII (X06DA), SLS | BM30, ESRF |
Detector | CMOS Photon 100 | PILATUS 2M-F | ADSC Q315r CCD |
Wavelength (Å) | 1.5418 | 1.0 | 0.979 |
a (Å) | 78.9 | 78.8 | 79.1 |
b (Å) | 88.8 | 89.1 | 89.1 |
c (Å) | 93.1 | 89.7 | 94.8 |
Space group | P212121 | P212121 | P212121 |
Molecules per ASU | 2 | 2 | 2 |
Matthews coefficient (Å3.Da−1) | 2.17 | 2.10 | 2.23 |
Resolution range (Å) | 23.81–1.80 (1.85–1.80) | 39.90–1.40 (1.42–1.40) | 65.00–2.30 (2.38–2.30) |
<I/σI> | 10.3 (2.31) | 11.4 (2.1) | 9.2 (2.2) |
Wilson B-factor | 14.5 | 12.7 | 27.4 |
Rmerge (%) * | 15.7 (62.6) | 6.8 (65.6) | 11.7 (72.3) |
Rpim (%) † | - | 4.2 (44.9) | 8.0 (48.7) |
Half-dataset correlation CC1/2 | - | 0.999 (0.813) | 0.995 (0.762) |
Multiplicity | 10 (7.8) | 6.4 (5.6) | 5.6 (5.6) |
No. of observed reflections | 588,364 | 797,508 (33,769) | 169,921 (16,295) |
No. of unique reflections | 61,589 | 124,619 (6083) | 30,270 (2907) |
Completeness (%) | 98.8 (99.9) | 99.9 (99.3) | 97.7 (94.9) |
Model Building and Refinement | |||
No. of protein atoms | 5080 | 5096 | 5032 |
No. of water molecules | 535 | 577 | 191 |
Rwork ‡ (%) | 21.9 | 19.6 | 18.0 |
Rfree § (%) | 25.5 | 22.1 | 23.3 |
R.m.s.d. bond lengths (Å) | 0.029 | 0.012 | 0.015 |
R.m.s.d. bond angles (⁰) | 2.810 | 1.972 | 2.124 |
Average B-factor (Å2) | |||
Protein | |||
Main-chain (A, B) | 20.1, 19.2 | 17.3, 16.3 | 37.8, 30.5 |
Side-chain (A, B) | 22.5, 21.4 | 20.6, 19.5 | 40.3, 33.5 |
Heme groups P heme_A, E heme_A P heme_B, E heme_B | 12.1, 14.9, 13.0, 13.7 | 11.0, 15.0 11.0, 12.0 | 25.2, 36.8 22.9, 21.1 |
Iron atoms P heme_A, E heme_A P heme_B, E heme_B | 13.0, 16.5, 13.9, 15.1 | 11.5, 14.8 11.2, 12.4 | 24.2, 36.7 21.9, 24.3 |
Calcium ions (A, B) | 11.7, 10.5 | 12.8, 10.4 | 28.3, 22.1 |
Water molecules | 25.9 (535) | 26.6 (577) | 30.1 (191) |
Azide ions (A, B) | - | - | 39.8, 51.3 |
Ramachandran plot | |||
Residues in favored regions (%) | 96.1 | 96.8 | 96.1 |
Residues in allowed regions (%) | 2.9 | 2.3 | 2.8 |
Residues outliers (%) | 0.9 | 0.9 | 1.1 |
PDB accession codes | 6FU3 | 7ZS8 | 6QKN |
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Nóbrega, C.S.; Carvalho, A.L.; Romão, M.J.; Pauleta, S.R. Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase—Insights into the Catalytic Cycle of Bacterial Peroxidases. Int. J. Mol. Sci. 2023, 24, 6246. https://doi.org/10.3390/ijms24076246
Nóbrega CS, Carvalho AL, Romão MJ, Pauleta SR. Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase—Insights into the Catalytic Cycle of Bacterial Peroxidases. International Journal of Molecular Sciences. 2023; 24(7):6246. https://doi.org/10.3390/ijms24076246
Chicago/Turabian StyleNóbrega, Cláudia S., Ana Luísa Carvalho, Maria João Romão, and Sofia R. Pauleta. 2023. "Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase—Insights into the Catalytic Cycle of Bacterial Peroxidases" International Journal of Molecular Sciences 24, no. 7: 6246. https://doi.org/10.3390/ijms24076246
APA StyleNóbrega, C. S., Carvalho, A. L., Romão, M. J., & Pauleta, S. R. (2023). Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase—Insights into the Catalytic Cycle of Bacterial Peroxidases. International Journal of Molecular Sciences, 24(7), 6246. https://doi.org/10.3390/ijms24076246