Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase)
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. Enzyme Assays
2.3. Homology Modeling
2.4. Site-Directed Mutagenesis
2.5. Long Inverse PCR
2.6. Molecular Docking
2.7. Statistics
3. Results
3.1. Characterization of G. stearothermophilus Hydantoinase
3.2. Homology Modeling of Thermostable D-Hydantoinase
3.3. Substrate Specificity and Kinetic Constants of Engineered Mutants of D-Hydantoinase
3.4. D-Hydantoinase Is the Enzyme of the Reductive Catabolism of Pyrimidines in G. stearothermophilus
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
GenBank Accession Number
References
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Divalent Metal Ion | Specific Enzyme Activity, U/mg | |
---|---|---|
0.2 mM | 2 mM | |
Mn2+ | 8.37 | 4.49 |
Fe2+ | 1.76 | 1.76 |
Co2+ | 1.76 | ND |
Ni2+ | 0.66 | ND |
Mg2+ | 0.61 | 1.26 |
Cu2+ | 0.55 | 1.56 |
Zn2+ | 0.55 | ND |
EDTA | 0.35 | 0.35 |
Substrate | D-Carbamoyl Methionine, mM |
---|---|
D, L-5-(2-methylthioethyl) hydantoin | 48.9 ± 2.1 |
D-5-2-methylthioethyl) hydantoin | 98.3 ± 2.5 |
L-5-(2-methylthioethyl) hydantoin | 1.6 ± 0.5 |
Mutation | Hydantoin | Dihydrouracil | D,L-5-methyl-lhydantoin | D,L-5-(2-methyl-thioethyl)-hydantoin | D,L-5-benzyl-hydantoin | D,L-5-indolylmethyl Hydantoin |
---|---|---|---|---|---|---|
Wild type | 3.5 ± 0.30 | 4.0 ± 0.31 | 1.4 ± 0.10 | 1.2 ± 0.09 | < 0.04 | < 0.04 |
W287A | 0.05 ± 0.01 | 0.43 ± 0.03 | 0.17 ± 0.02 | 0.54 ± 0.04 | 1.75 ± 0.12 | 1.57 ± 0.12 |
F159A | 0.06 ± 0.01 | 5.50 ± 0.40 | < 0.04 | 0.58 ± 0.04 | 1.30 ± 0.10 | 1.10 ± 0.08 |
W287A/F159A | 0.32 ± 0.02 | 0.85 ± 0.07 | 0.53 ± 0.03 | 0.08 ± 0.01 | 1.75 ± 0.15 | 1.16 ± 0.09 |
I190A | 0.56 ± 0.04 | 2.2 ± 0.10 | 0.22 ± 0.01 | 0.53 ± 0.04 | 0.05 ± 0.01 | < 0.04 |
R212K | 1.19 ± 0.10 | 0.06 ± 0.01 | 0.22 ± 0.01 | 0.5 ± 0.04 | < 0.04 | < 0.04 |
W287A/R212K | 1.1 ± 0.10 | < 0.04 | < 0.04 | 0.27 ± 0.02 | 0.5 ± 0.04 | < 0.04 |
Enzyme | Km, mM | kcat/Km, (s·mM) −1 | kcat, s−1 | |||
---|---|---|---|---|---|---|
Dihydrouracil | D,L-5-indolylmethyl Hydantoin | Dihydrouracil | D,L-5-indolyl-methyl Hydantoin | Dihydrouracil | D,L-5-indolyl-methyl hydantoin | |
Wild | 0.97 ± 0.22 | ND | 11.3 ± 2.20 | ND | 11.2 ± 0.70 | ND |
W287A | 0.43 ± 0.13 | 1.66 ± 0.85 | 6.53 ± 1.83 | 4.13 ± 1.63 | 2.76 ± 0.17 | 6.83 ± 0.97 |
F159A | 2.89 ± 1.27 | 0.60 ± 0.58 | 7.53 ± 2.50 | 6.89 ± 5.83 | 21.8 ± 2.60 | 4.16 ± 0.60 |
W287A/F159A | 5.61 ± 2.32 | 4.30 ± 0.40 | 1.10 ± 0.30 | 2.90 ± 0.17 | 6.16 ± 1.00 | 12.5 ± 0.50 |
I190A | 1.12 ± 0.61 | ND | 7.09 ± 3.20 | ND | 7.99 ± 0.90 | ND |
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Aganyants, H.; Weigel, P.; Hovhannisyan, Y.; Lecocq, M.; Koloyan, H.; Hambardzumyan, A.; Hovsepyan, A.; Hallet, J.-N.; Sakanyan, V. Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase). High-Throughput 2020, 9, 5. https://doi.org/10.3390/ht9010005
Aganyants H, Weigel P, Hovhannisyan Y, Lecocq M, Koloyan H, Hambardzumyan A, Hovsepyan A, Hallet J-N, Sakanyan V. Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase). High-Throughput. 2020; 9(1):5. https://doi.org/10.3390/ht9010005
Chicago/Turabian StyleAganyants, Hovsep, Pierre Weigel, Yeranuhi Hovhannisyan, Michèle Lecocq, Haykanush Koloyan, Artur Hambardzumyan, Anichka Hovsepyan, Jean-Noël Hallet, and Vehary Sakanyan. 2020. "Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase)" High-Throughput 9, no. 1: 5. https://doi.org/10.3390/ht9010005
APA StyleAganyants, H., Weigel, P., Hovhannisyan, Y., Lecocq, M., Koloyan, H., Hambardzumyan, A., Hovsepyan, A., Hallet, J.-N., & Sakanyan, V. (2020). Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase). High-Throughput, 9(1), 5. https://doi.org/10.3390/ht9010005