Del Arco, JonMills, AlbertoGago, FedericoFernández Lucas, Jesús2024-11-052024-11-052019Del Arco, J., Mills, A., Gago, F., & Fernández-Lucas, J. (2019). Structure-Guided Tuning of a Selectivity Switch towards Ribonucleosides in Trypanosoma brucei Purine Nucleoside 2′-Deoxyribosyltransferase. ChemBioChem, 20(24), 2996-3000. https://doi.org/10.1002/CBIC.2019003971439-422710.1002/cbic.201900397https://hdl.handle.net/20.500.14352/110035The use of nucleoside 2′-deoxyribosyltransferases (NDTs) as biocatalysts for the industrial synthesis of nucleoside analogues is often hindered by their strict preference for 2′-deoxyribonucleosides. It is shown herein that a highly versatile purine NDT from Trypanosoma brucei (TbPDT) can also accept ribonucleosides as substrates; this is most likely because of the distinct role played by Asn53 at a position that is usually occupied by Asp in other NDTs. Moreover, this unusual activity was improved about threefold by introducing a single amino acid replacement at position 5, following a structure-guided approach. Biophysical and biochemical characterization revealed that the TbPDTY5F variant is a homodimer that displays maximum activity at 50 °C and pH 6.5 and shows a remarkably high melting temperature of 69 °C. Substrate specificity studies demonstrate that 6-oxopurine ribonucleosides are the best donors (inosine>guanosine≫adenosine), whereas no significant preferences exist between 6-aminopurines and 6-oxopurines as base acceptors. In contrast, no transferase activity could be detected on xanthine and 7-deazapurines. TbPDTY5F was successfully employed in the synthesis of a wide range of modified ribonucleosides containing different purine analogues.engjournal article1439-7633https://doi.org/10.1002/cbic.201900397https://chemistry-europe.onlinelibrary.wiley.com/doi/epdf/10.1002/cbic.201900397restricted access577.113.3577.15577.266.0BiocatalysisEnzymesImmobilizationMolecular dynamicsNucleosidesBioquímica (Biología)Biología molecular (Biología)Química industrial2403 Bioquímica2415 Biología Molecular2302.09 Enzimología2302.23 Ácidos Nucleicos