Agaricales Mushroom Lignin Peroxidase: From Structure–Function to Degradative Capabilities
Abstract
:1. Introduction
2. Materials and Methods
2.1. Gene Synthesis and Site-Directed Mutagenesis
2.2. Heterologous Expression and In Vitro Activation
2.3. Protein Purification and Quantification
2.4. ApeLiP Crystallization, Data Collection, and Refinement
2.5. pH and Temperature Stability
2.6. Steady-State Kinetic Constants
2.7. Native and Acetylated Softwood and Hardwood Lignins
2.8. Estimation of Transient-State Kinetic Constants with Lignosulfonates
2.9. Estimation of CI/RS, CI/CII and CII/RS Reduction Potentials
2.10. Steady-State Treatment of Lignin
2.11. 2D-NMR Analyses
3. Results
3.1. Heterologous Expression, Protein Activation, and Purification of ApeLiP
3.2. ApeLiP Crystallographic Studies
3.3. Temperature and pH Stability of Recombinant ApeLiP
3.4. Steady-State Kinetic Properties of ApeLiP
3.5. Identification of Trp166 as a Catalytic Residue
3.6. Catalytic Cycle and ApeLiP Reduction Potentials
3.7. Lignin Oxidation by ApeLiP: Transient-State Kinetic Data and NMR Analyses
4. Discussion
4.1. Lignin Peroxidase in Agaricales Genomes
4.2. ApeLiP Activation and LRET Oxidation of Lignin Models
4.3. Heme-Channel Oxidation Site
4.4. Intriguing Catalytic Cycle and Lignin Decay Abilities
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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Data Collection | Refinement | ||
---|---|---|---|
Resolution range (Å) | 74.95–1.85 (1.95–1.85) | Resolution (Å) | 55.56–1.85 |
Space group | P 21 21 21 | Working reflections | 26,256 (2733) |
Unit cell (Å) | a = 48.13, b = 74.95, c = 82.77 | Reflections test set | 1270 (140) |
Total reflections | 164,812 (23,933) | R-work (%) | 22.6 (40.8) |
Unique reflections | 26,316 (3760) | R-free (%) | 26.8 (42.1) |
Multiplicity | 6.3 (6.4) | Number of non-H atoms | |
Completeness (%) | 99.8 (100.0) | - protein | 2413 |
Mean I/sigma (I) | 15.5 (3.0) | - heme group | 43 |
Wilson B-factor (Å2) | 35.8 | - ions | 4 |
Rmerge | 0.051 (0.483) | - solvent | 83 |
Rmeas | 0.061 (0.575) | Average B-factor (Å2) | |
Rpim | 0.033 (0.309) | - protein | 48.02 |
CC1/2 | 0.99 (0.93) | - heme group | 33.78 |
- ions | 43.51 | ||
- solvent | 45.56 | ||
rmsd bond lengths (Å) | 0.012 | ||
rmsd angles (°) | 1.67 | ||
Ramachandran statistics | |||
- Preferred (%) | 92.71 | ||
- Allowed (%) | 5.78 | ||
- Outliers (%) | 1.52 | ||
PDB code | 7OO5 |
ABTS a | DMP | |||||||
---|---|---|---|---|---|---|---|---|
H2O2 | Low Efficiency | High Efficiency | pH 3 | pH8 | VA | RB5 | ||
ApeLiP | KM | 33 ± 4.4 | 693 ± 63 | 8.1 ± 0.6 b | 37 ± 5.6 | 1430 ± 440 | (29.6 ± 4.6) × 103 | 14 ± 1.4 b |
kcat | 100 ± 4.8 | 179 ± 5 | 42 ± 1.8 | 68.6 ± 2.8 | 5.7 ± 0.7 | 4.3 ± 0.3 | 21 ± 1.5 | |
kcat/KM | 3070 ± 440 | 258 ± 25 | 5130 ± 460 | 1910 ± 310 | 4.0 ± 1.3 | 0.15 ± 0.03 | 1540 ± 115 | |
ki | (2.1 ± 0.5) × 103 | - | - | (11 ± 2.2) × 103 | nd d | (406 ± 92) × 103 | - | |
W166A | KM | 136 ± 12 | 1150 ± 190 c | - | - | 49 ± 8.2 | - | - |
kcat | 126 ± 4.7 | 117 ± 10 c | - | - | 6.1 ± 0.27 | - | - | |
kcat/KM | 929 ± 87 | 102 ± 19 c | - | - | 125 ± 22 | - | - | |
ki | (3.3 ± 0.3) × 103 | nd c | - | - | nd d | - | - |
Softwood Lignin | Hardwood Lignin | ||||
---|---|---|---|---|---|
Native | Acetylated | Native | Acetylated | ||
ApeLiP | KD3 | ns b | 31 ± 6.9 | 156 ± 25 | 89 ± 14 |
k3 | ns | 26 ± 1.7 | 163 ± 38 | 56 ± 4.6 | |
k3app | 926 ± 47 | 830 ± 195 | 957 ± 270 | 630 ± 111 | |
W166A | KD3 | - | - | - | - |
k3 | - | - | - | - | |
k3app | 0 | 0 | 0 | 0 | |
P. eryngii VPL d | KD3 | 143 ± 19 | 24 ± 1.9 | 14 ± 1 | 21 ± 2.5 |
k3 | 48 ± 2 | 14 ± 0.4 | 14 ± 2 | 12 ± 0.5 | |
k3app | 340 ± 30 | 599 ± 31 | 990 ± 80 | 592 ± 52 | |
P. chrysosporium LiPA e | KD3 | 95 ± 26 | na c | 19 ± 2 | na |
k3 | 25 ± 4 | na | 14 ± 0 | na | |
k3app | 263 ± 83 | na | 764 ± 86 | na |
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Sánchez-Ruiz, M.I.; Ayuso-Fernández, I.; Rencoret, J.; González-Ramírez, A.M.; Linde, D.; Davó-Siguero, I.; Romero, A.; Gutiérrez, A.; Martínez, A.T.; Ruiz-Dueñas, F.J. Agaricales Mushroom Lignin Peroxidase: From Structure–Function to Degradative Capabilities. Antioxidants 2021, 10, 1446. https://doi.org/10.3390/antiox10091446
Sánchez-Ruiz MI, Ayuso-Fernández I, Rencoret J, González-Ramírez AM, Linde D, Davó-Siguero I, Romero A, Gutiérrez A, Martínez AT, Ruiz-Dueñas FJ. Agaricales Mushroom Lignin Peroxidase: From Structure–Function to Degradative Capabilities. Antioxidants. 2021; 10(9):1446. https://doi.org/10.3390/antiox10091446
Chicago/Turabian StyleSánchez-Ruiz, María Isabel, Iván Ayuso-Fernández, Jorge Rencoret, Andrés Manuel González-Ramírez, Dolores Linde, Irene Davó-Siguero, Antonio Romero, Ana Gutiérrez, Angel T. Martínez, and Francisco Javier Ruiz-Dueñas. 2021. "Agaricales Mushroom Lignin Peroxidase: From Structure–Function to Degradative Capabilities" Antioxidants 10, no. 9: 1446. https://doi.org/10.3390/antiox10091446