Structural Characterization of Two Short Unspecific Peroxygenases: Two Different Dimeric Arrangements
Abstract
:1. Introduction
2. Materials and Methods
2.1. Protein Expression and Purification
2.2. Site-Directed Mutagenesis
2.3. Crystallization, Data Collection, and Processing
2.4. Structure Determination and Refinement
2.5. Size-Exclusion Chromatography and Analytical Ultracentrifugation
2.6. Enzyme Kinetics
2.7. Fatty Acid Enzymatic Oxygenation
3. Results
3.1. Production of Native UPOs and Site-Directed Variants
3.2. Overall Crystallographic Structures
3.3. Heme Pocket
3.4. Dimeric Arrangements
3.5. Biophysical Properties: Molecular-Mass and Oligomerization State
3.6. Comparison of Catalytic Properties
4. Discussion
4.1. Short and Long UPO Families
4.2. Two Different Dimeric Arrangements
4.3. Some (Bio)Technological Implications
4.4. Concluding Remarks
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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rMroUPO | rCviUPO | |
---|---|---|
Wavelength (Å) | 0.9792 | 0.9792 |
Space group | C2221 | P212121 |
Unit cell dimensions: | ||
a | 100.3 Å | 78.5 Å |
b | 107.4 Å | 140.1 Å |
c | 186.2 Å | 42.2 Å |
Resolution (Å) | 73.3–1.45 | 70.1–1.95 |
Rmeas a | 0.094 (0.81) | 0.191 (0.99) |
Rpim | 0.037 (0.33) | 0.057 (0.40) |
I/σI | 12.0 (2.3) | 10.1 (2.4) |
Completeness (%) | 99.4 (99.5) | 100 (100) |
Redundancy | 6.1 (5.8) | 10.6 (6.2) |
CC1/2 b (%) | 99.7 (66.2) | 99.4 (83.5) |
rMroUPO | rCviUPO | |
---|---|---|
Refinement: | ||
Resolution (Å) | 73.3–1.45 | 70.1–1.95 |
No. unique reflections | 167,101 (12,200) | 33,334 (2439) |
Rwork/Rfree | 0.158/0.185 | 0.191/0.243 |
No. atoms: | ||
Protein | 7276 | 3664 |
Ligand | 172 | 86 |
Ion | 4 | 2 |
Water | 1066 | 268 |
B-factors (Å2): | ||
Protein | 15.7 | 26.0 |
Ligand | 12.6 | 20.3 |
Ion | 8.38 | 14.97 |
Water | 28.54 | 34.1 |
R.m.s deviations: | ||
Bond lengths (Å) | 0.013 | 0.010 |
Bond angles (°) | 1.85 | 1.53 |
Ramachandran: | ||
Favored (%) | 96.41 | 94.21 |
Allowed (%) | 3.17 | 5.57 |
Outliers (%) | 0.42 | 0.22 |
PDB codes: | 7ZBP | 7ZCL |
MroUPO | rMroUPO | C227A | rCviUPO | C235A | K228stop | ||
---|---|---|---|---|---|---|---|
Veratryl alcohol | Km | 279 | 54.2 ± 16.7 | - | 2940 ± 160 | 7240 ± 820 | 55,300 ± 10,000 |
kcat | 49 | 2.49 ± 0.16 | 0 | 2.24 ± 0.03 | 11.2 ± 0.4 | 56.4 ± 6.6 | |
kcat/Km | 176 | 46 ± 12 | - | 0.75 ± 0.03 | 1.5 ± 0.2 | 1.0 ± 0.2 | |
ABTS | Km | 71 | 246 ± 3 | - | 239 ± 8 1 | 87 ± 7 | 110 ± 15 |
kcat | 25 | 15.0 ± 3.3 | 0 | 157.2 ± 2.8 | 257.0 ± 4.9 | 209 ± 7 | |
kcat/Km | 350 | 62 ± 14 | - | 656 ± 26 | 2970 ± 190 | 1900 ± 210 | |
DMP | Km | 133 | 206 ± 38 | - | 4930 ± 470 | 4500 ± 424 | 3720 ± 250 |
kcat | 70 | 66.2 ± 3.1 | 0 | 325 ± 11 | 812 ± 26 | 728 ± 16 | |
kcat/Km | 530 | 320 ± 50 | - | 66 ± 5 | 180 ± 12 | 195 ± 10 | |
H2O2 | Km | 3140 | 1880 ± 130 | - | 2250 ± 750 2 | 420 ± 6 3 | 680 ± 120 4 |
kcat/Km | 24.2 | 25.8 ± 0.4 | - | 1120 ± 475 | 5170 ± 130 | 3930 ± 170 |
Conversion | Products | (%) | Epoxidation | ||||
---|---|---|---|---|---|---|---|
(%) | 12-Epoxy | 9-Epoxy | Diepoxy | Hydroxy | OH-Epoxy | Yield (%) | |
rCviUPO | 97 | 56 | 10 | - | 8 | 26 | 45 |
K228stop | 98 | 14 | 16 | 51 | 13 | 6 | 68 |
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Linde, D.; Santillana, E.; Fernández-Fueyo, E.; González-Benjumea, A.; Carro, J.; Gutiérrez, A.; Martínez, A.T.; Romero, A. Structural Characterization of Two Short Unspecific Peroxygenases: Two Different Dimeric Arrangements. Antioxidants 2022, 11, 891. https://doi.org/10.3390/antiox11050891
Linde D, Santillana E, Fernández-Fueyo E, González-Benjumea A, Carro J, Gutiérrez A, Martínez AT, Romero A. Structural Characterization of Two Short Unspecific Peroxygenases: Two Different Dimeric Arrangements. Antioxidants. 2022; 11(5):891. https://doi.org/10.3390/antiox11050891
Chicago/Turabian StyleLinde, Dolores, Elena Santillana, Elena Fernández-Fueyo, Alejandro González-Benjumea, Juan Carro, Ana Gutiérrez, Angel T. Martínez, and Antonio Romero. 2022. "Structural Characterization of Two Short Unspecific Peroxygenases: Two Different Dimeric Arrangements" Antioxidants 11, no. 5: 891. https://doi.org/10.3390/antiox11050891
APA StyleLinde, D., Santillana, E., Fernández-Fueyo, E., González-Benjumea, A., Carro, J., Gutiérrez, A., Martínez, A. T., & Romero, A. (2022). Structural Characterization of Two Short Unspecific Peroxygenases: Two Different Dimeric Arrangements. Antioxidants, 11(5), 891. https://doi.org/10.3390/antiox11050891