Identification of a 2′-O-Methyluridine Nucleoside Hydrolase Using the Metagenomic Libraries
Abstract
:1. Introduction
2. Results
2.1. Discovery of RK9 Nucleoside Hydrolase and Deoxyribose-Phosphate Aldolase
2.2. Activity of RK9NH and RK9DPA Proteins in E. coli Cells
2.3. Substrate Specificity of RK9NH Protein
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains, Plasmids, Primers, Media and Reagents
4.2. The E. coli Uracil Auxotroph Strain and the Metagenomic Libraries
4.3. Synthesis of 5-fluoro-2′-O-methyluridine
4.4. Over-Expression and Purification of the Recombinant RK9NH Protein
4.5. Substrate Specificity Measurements
4.6. Thin-Layer Chromatography (TLC)
4.7. HPLC-MS
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Samples of the plasmids created in this work are available from the authors on request. |
Strain | Carbon Source | ||
---|---|---|---|
None | Glucose | Thymidine | |
E. coli BW25113 | - | ++ | + |
E. coli BW25113 deoC::kan | - | ++ | - |
E. coli BW25113 deoC::kan pQE70-RK9DPA | - | ++ | - |
Substrate | Conversion (%) |
---|---|
Uridine | 100 |
2′-Deoxyuridine | 0 |
2′-O-methyluridine | 100 |
3′-O-methyluridine | 0 |
2′-O-allyluridine | 0 |
3′-O-allyluridine | 0 |
5-Fluorouridine | 100 |
5-Fluoro-2′-deoxyuridine | 4 ± 0.1 |
5-Fluoro-2′-O-methyluridine | 100 |
2′-Amino-2′-deoxyuridine | 0 |
Cytidine | 65 ± 1 |
2′-Deoxycytidine | 0 |
2′-O-methylcytidine | 70 ± 1 |
5-Methyluridine | 100 |
Thymidine | 0 |
Adenosine | 10 ± 0.2 |
2′-Deoxyadenosine | 0 |
2′-O-methyladenosine | 5 ± 0.1 |
Guanosine | 100 |
2′-Deoxyguanosine | 3 ± 0.1 |
2′-O-methylguanosine | 5 ± 0.1 |
Inosine | 22 ± 0.5 |
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Aučynaitė, A.; Rutkienė, R.; Tauraitė, D.; Meškys, R.; Urbonavičius, J. Identification of a 2′-O-Methyluridine Nucleoside Hydrolase Using the Metagenomic Libraries. Molecules 2018, 23, 2904. https://doi.org/10.3390/molecules23112904
Aučynaitė A, Rutkienė R, Tauraitė D, Meškys R, Urbonavičius J. Identification of a 2′-O-Methyluridine Nucleoside Hydrolase Using the Metagenomic Libraries. Molecules. 2018; 23(11):2904. https://doi.org/10.3390/molecules23112904
Chicago/Turabian StyleAučynaitė, Agota, Rasa Rutkienė, Daiva Tauraitė, Rolandas Meškys, and Jaunius Urbonavičius. 2018. "Identification of a 2′-O-Methyluridine Nucleoside Hydrolase Using the Metagenomic Libraries" Molecules 23, no. 11: 2904. https://doi.org/10.3390/molecules23112904