Arco Arrieta, Jon delMills, AlbertoGago, FedericoFernández Lucas, Jesús2022-02-212022-02-212019Arco, 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.2019003971439-42271439-7633http://hdl.handle.net/11268/10788The 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.engNucleósidosSimulación de dinámica molecularjournal article10.1002/cbic.201900397restricted accessEstructura molecularEnzimaInvestigación química