Gene

Deoxyguanosine

Purine

Inosine

Biochemistry

Purine nucleoside phosphorylase

Stereochemistry

Combinatorial chemistry

Hypoxanthine

Adenosine

Nucleotide

Nucleoside

Enzyme

Guanosine

Chemistry

Trypanosoma brucei

ChemBioChem

The 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 ( Tb PDT) 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 Tb PDT Y5F 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&gt;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. Tb PDT Y5F was successfully employed in the synthesis of a wide range of modified ribonucleosides containing different purine analogues.

Structure‐Guided Tuning of a Selectivity Switch towards Ribonucleosides in <i>Trypanosoma brucei</i> Purine Nucleoside 2′‐Deoxyribosyltransferase

Structure‐Guided Tuning of a Selectivity Switch towards Ribonucleosides in Trypanosoma brucei Purine Nucleoside 2′‐Deoxyribosyltransferase