Structural and Mechanistic Insights into a Novel Monooxygenase for Poly(acrylic acid) Biodegradation
Abstract
:1. Introduction
2. Results and Discussion
2.1. Bioinformatic Analyses of Monooxygenase PCX02514 with PAA Degradation Ability
2.2. Biochemical Analyses of Monooxygenase PCX02514
2.3. Crystal Structure of Monooxygenase PCX02514
2.4. Active Sites of Monooxygenase PCX02514 Binding with Substrates
3. Materials and Methods
3.1. Bacterial Strain, Culture Medium, and Chemicals
3.2. Bioinformatic Analyses
3.3. Cloning, Expression, and Purification
3.4. HPLC–MS Analysis
3.5. Gel Permeation Chromatography Analysis
3.6. Enzyme Kinetic Analysis
3.7. Isothermal Titration Calorimetry
3.8. Crystallization, Data Collection, and Structure Determination
3.9. Molecular Docking of Monooxygenase PCX02514 and Substrates
3.10. Site-Directed Mutagenesis and Enzyme Activity Measurement
3.11. Statistical Analysis
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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KM (μM) | Vm (μM s−1) | kcat (s−1) | kcat/KM (s−1 M−1) |
---|---|---|---|
158.9 ± 16.4 | 0.5376 ± 0.1284 | 0.00214 ± 0.000511 | 13.47 |
N (Sites) | KD (×10−9 M) | ΔH (kcal mol−1) | ΔS (cal mol−1 K−1) | ΔG (kcal mol−1) | −TΔS (kcal mol−1) |
---|---|---|---|---|---|
0.034 ± 0.0027 | 239 ± 15.7 | −32.9 ± 5.42 | −0.08 ± 0.013 | −9.04 ± 1.63 | 23.9 ± 3.79 |
Protein | PCX02514 |
---|---|
PDB accession | 8K74 |
Data collection | |
Beamline | BL18U1 |
Wavelength (Å) | 0.9870 |
Space group | P 1 21 1 |
Cell dimensions | |
a, b, c (Å) | 73.23, 59.61, 79.72 |
α, β, γ (°) | 90.00, 98.84, 90.00 |
Resolution (Å) | 41.54–1.97(2.02–1.97) a |
No. observed reflections | 44,451 (2618) |
No. unique reflections | 44,419 (2618) |
Rmerge (%) | 10.6 (68.0) |
I/σI | 8.2 (3.15) |
Completeness (%) | 92.7 (78.3) |
Redundancy | 1.0 (1.0) |
CC1/2 (%) | 0.0 |
Refinement | |
Resolution (Å) | 41.54–1.97 |
Rwork/Rfree | 0.183/0.222 |
No. atoms | 5201 |
Protein | 4932 |
Ligand/ion | 0 |
Water | 269 |
B-factors | 24.20 |
Protein | 25.79 |
Ligand/ion | 0 |
Water | 27.76 |
R.m.s. deviations | |
Bond lengths (Å) | 0.007 |
Bond angles (°) | 0.942 |
Ramachandran plot (%) | |
Favored | 97.83 |
Allowed | 1.86 |
Disallowed | 0.31 |
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Feng, R.; Zhao, J.; Li, X.; Dong, S.; Ma, D. Structural and Mechanistic Insights into a Novel Monooxygenase for Poly(acrylic acid) Biodegradation. Int. J. Mol. Sci. 2024, 25, 8871. https://doi.org/10.3390/ijms25168871
Feng R, Zhao J, Li X, Dong S, Ma D. Structural and Mechanistic Insights into a Novel Monooxygenase for Poly(acrylic acid) Biodegradation. International Journal of Molecular Sciences. 2024; 25(16):8871. https://doi.org/10.3390/ijms25168871
Chicago/Turabian StyleFeng, Rui, Juyi Zhao, Xiaochen Li, Sijun Dong, and Dan Ma. 2024. "Structural and Mechanistic Insights into a Novel Monooxygenase for Poly(acrylic acid) Biodegradation" International Journal of Molecular Sciences 25, no. 16: 8871. https://doi.org/10.3390/ijms25168871