| Autor: |
Hormigo D; Applied Biotechnology Group, Universidad Europea de Madrid, Urbanización El Bosque, Villaviciosa de Odón, 28670 Madrid, Spain., Del Arco J; Applied Biotechnology Group, Universidad Europea de Madrid, Urbanización El Bosque, Villaviciosa de Odón, 28670 Madrid, Spain., Acosta J; Applied Biotechnology Group, Universidad Europea de Madrid, Urbanización El Bosque, Villaviciosa de Odón, 28670 Madrid, Spain., Fürst MJLJ; Molecular Enzymology Group, University of Groningen, Feringa Building, 9747 AG Groningen, The Netherlands., Fernández-Lucas J; Applied Biotechnology Group, Universidad Europea de Madrid, Urbanización El Bosque, Villaviciosa de Odón, 28670 Madrid, Spain.; Grupo de Investigación en Ciencias Naturales y Exactas, GICNEX, Universidad de la Costa, CUC, Calle 58 #55-66, Barranquilla 080002, Colombia.; Department of Biochemistry and Molecular Biology, Faculty of Biology, Universidad Complutense de Madrid, C. de José Antonio Novais, 12, 28040 Madrid, Spain. |
| Abstrakt: |
Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis-unlike pyrimidine NPs (PyNPs, UP, and TP)-multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus . These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60-90 °C) and within a pH range of 6-8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80-100% of their activity when incubated at 60-80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2'-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2'-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis. |
