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Abstract
AbstractThe use of enzymes for the synthesis of nucleoside analogues offers several advantages over multistep chemical methods, including chemo‐, regio‐ and stereoselectivity as well as milder reaction conditions. Herein, the production, characterization and utilization of a purine nucleoside 2’‐deoxyribosyltransferase (PDT) from Trypanosoma brucei are reported. TbPDT is a dimer which displays not only excellent activity and stability over a broad range of temperatures (50–70 °C), pH (4–7) and ionic strength (0–500 mM NaCl) but also an unusual high stability under alkaline conditions (pH 8–10). TbPDT is shown to be proficient in the biosynthesis of numerous therapeutic nucleosides, including didanosine, vidarabine, cladribine, fludarabine and nelarabine. The structure‐guided replacement of Val11 with either Ala or Ser resulted in variants with 2.8‐fold greater activity. TbPDT was also covalently immobilized on glutaraldehyde‐activated magnetic microspheres. MTbPDT3 was selected as the best derivative (4200 IU/g, activity recovery of 22 %), and could be easily recaptured and recycled for >25 reactions with negligible loss of activity. Finally, MTbPDT3 was successfully employed in the expedient synthesis of several nucleoside analogues. Taken together, our results support the notion that TbPDT has good potential as an industrial biocatalyst for the synthesis of a wide range of therapeutic nucleosides through an efficient and environmentally friendly methodology.
Highlights
TbPDT has good potential as an industrial biocatalyst for the synthesis of a wide range of therapeutic nucleosides through an efficient and environmentally friendly methodology
In our search for purine-nucleoside 2’-deoxyribosyltransferases (PDTs) suitable as biocatalysts for the synthesis of non-natural therapeutic nucleosides, we found a good candidate in an uncharacterized protein encoded in the genome of Trypanosoma brucei (TbPDT) that shares 67 %
We report the cloning of the pdt gene, its expression in E. coli, and the purification and biochemical characterization of the encoded protein, TbPDT
Summary
TbPDT has good potential as an industrial biocatalyst for the synthesis of a wide range of therapeutic nucleosides through an efficient and environmentally friendly methodology. Nucleoside 2’-deoxyribosyltransferases (NDTs) have been generally employed in the synthesis of nucleosides because they catalyze the exchange of 2’-deoxyribose between purine and/or pyrimidine bases.[3b,4] According to their substrate specificity, NDTs have been classified as type I, which transfer the glycosyl moiety from purine 2’-deoxyribonucleosides to purine bases (Pur$Pur) such as adenine 1, guanine 2 or hypoxanthine 3 (Figure 1), and type II, which accept pyrimidines as substrates (Pur$Pur, Pur$Pyr, Pyr$Pyr).[3b,4–5] For this reason, type I NDTs are known as purine-nucleoside 2’-deoxyribosyltransferases (PDTs).
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