Catabolic Reductive Dehalogenase Substrate Complex Structures Underpin Rational Repurposing of Substrate Scope
Abstract
:1. Introduction
2. Materials and Methods
2.1. Molecular Biology
2.2. Transformation Protocols
2.3. Heterologous Expression
2.4. Protein Purification
2.5. Cofactor Analysis
2.6. NpRdhA Activity Analysis
2.7. HPLC Analysis
2.8. Crystallogenesis
2.9. Diffraction Data Collection and Data Processing
2.10. EPR Spectroscopy
3. Results
3.1. Purification and Characterization of N-Terminally Hexa-Histidine Tagged NpRdhA
3.2. High-Resolution Crystal Structure of N-Terminally Tagged NpRdhA
3.3. Crystal Structure of NpRdhAN Ligand Complexes
3.4. Structure of NpRdhAN K488Q Variant
3.5. Rational Mutagenesis of NpRdhA
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Primer | Sequence |
---|---|
NpRdhAN-pPT7-F | GGAGGTGAAATGTACAATGGTCCAAACTAGTTTCGAACATCACCATCACCATCACTCAGCTGGTGAAAACTTATATTTCCAAGGCGCCCAGATCTCCATGCGCCTTTATTCCAATCG |
NpRdhAN-pPT7-R | GGCCGGTACCGGATCCTTAGCCAGCGCTAGATTTAAGAC |
NpRdhA-K488Q-F | TCCGTTTATTGGTCCTCGTTCCAAAAGTATCGTTTTTACAA |
NpRdhA-K488Q-R | TTGTAAAAACATACTTTTGGAACGAGGACCAATAAACGGA |
NpRdhA-A419M-F | AAGGAGCTTCAGGTGATGATTGGATTTCAATGAGTCAGTCCATGCGT |
NpRdhA-A419M-R | ACGCATGGACTGACTCATTGAAATCCAATCAT CACCTGAAGCTCCTT |
Structure (PDB Code) | NpRdhAN (6ZXU) | NpRdhAN:35-DB-4-OH (6ZXX) | NpRdhAN:3-B-4-OH (6ZY1) | NpRdhAN-K488Q (6ZY0) |
---|---|---|---|---|
Data Collection | ||||
Space Group | C 2 | C 2 | C 2 | C 2 |
a, b, c (Å) | 175.11 170.96 107.87 | 180.18 170.39 107.96 | 175.87 170.10 107.65 | 176.35 169.57 107.80 |
β (°) | 98.37 | 99.15 | 98.35 | 98.33 |
Resolution (Å) | 121.7–1.73 (1.91–1.73) | 123.1–1.99 (2.26–1.99) | 106.51–1.99 (2.21–1.99) | 106.66–2.13 (2.44–2.13) |
Rmeas | 0.104 (0.873) | 0.149 (0.886) | 0.181 (0.827) | 0.196 (1.238) |
I/σI | 8.7 (1.6) | 7.3 (1.8) | 6.8 (1.7) | 8.2 (1.8) |
Completeness (%) | 93.0 (58.0) | 94.5 (75.5) | 94.0 (78.5) | 94.5 (74.6) |
Redundancy | 3.3 (2.3) | 3.4 (3.2) | 3.5 (3.1) | 7.0 (6.7) |
Refinement | ||||
No. Reflections | 201,352 (685) | 97,499 (302) | 112,203 (229) | 84,097 (316) |
Rwork/Rfree | 0.1723/0.2026 (0.3239/0.3074) | 0.1812/0.2233 (0.3106/0.3295) | 0.1769/0.2180 (0.2354/0.2923) | 0.1841/0.2328 (0.3054/0.5585) |
Nr Atoms Protein | 15,975 | 16,128 | 15,902 | 16,508 |
Nr Atoms Solvent | 1703 | 696 | 968 | 305 |
Ion/Ligand | 15 | 21 | 15 | 12 |
Protein B-factor (Å2) | 27.1 | 31.7 | 23.7 | 36.0 |
Solvent B-factor (Å2) | 33.3 | 32.9 | 27.8 | 34.2 |
Rmsd Bond Length (Å) | 0.009 | 0.010 | 0.014 | 0.010 |
Rmsd Bond Angles (°) | 1.556 | 1.507 | 1.072 | 1.541 |
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Halliwell, T.; Fisher, K.; Payne, K.A.P.; Rigby, S.E.J.; Leys, D. Catabolic Reductive Dehalogenase Substrate Complex Structures Underpin Rational Repurposing of Substrate Scope. Microorganisms 2020, 8, 1344. https://doi.org/10.3390/microorganisms8091344
Halliwell T, Fisher K, Payne KAP, Rigby SEJ, Leys D. Catabolic Reductive Dehalogenase Substrate Complex Structures Underpin Rational Repurposing of Substrate Scope. Microorganisms. 2020; 8(9):1344. https://doi.org/10.3390/microorganisms8091344
Chicago/Turabian StyleHalliwell, Tom, Karl Fisher, Karl A. P. Payne, Stephen E. J. Rigby, and David Leys. 2020. "Catabolic Reductive Dehalogenase Substrate Complex Structures Underpin Rational Repurposing of Substrate Scope" Microorganisms 8, no. 9: 1344. https://doi.org/10.3390/microorganisms8091344