Impact of 6-purine modifications on the ribose substrate selectivity of pyrimidine nucleoside phosphorylase (PyNP) in base exchange reactions

Pyrimidine nucleoside phosphorylase (PyNP) catalyzes phosphorolysis and base exchange reactions, providing an eco-friendly and stereoselective approach for synthesizing unnatural nucleosides. This study investigates PyNP substrate specificity, particularly the effects of functional group modifications at the 6-position of purine nucleobases on enzymatic base exchange reactions using thymidine (Thd) or uridine (Urd) as ribose donors. The reaction rate with the two ribose donors varied significantly depending on the 6-substituent on purine bases. Compounds with 6-monoalkylamino groups preferentially reacted with uridine (Thd/Urd values lower than 1.0), while 6-dialkylamino groups favored thymidine (Thd/Urd values greater than 1). When an ether or alkyl group was introduced at the 6-position, the corresponding purine was found to react preferentially with thymidine as a ribose donor. Docking simulations suggested that the steric hindrance of a 6-dialkylamino group affected substrate positioning, leading to a preferential reaction with thymidine, while increasing the steric hindrance of the 2'-hydroxyl group of uridine. On the other hand, 6-monoalkylaminopurines were less sterically hindered, and the ribose moiety was located away from the pocket surface of the enzyme, suggesting that the presence or absence of the 2'-hydroxyl group of the ribose form had minimal impact. Thus, selectivity (Thd/Urd value) showed a slight preference for uridine, albeit not significant. These results demonstrate that, in the bi-substrate enzymatic reaction of PyNP, one substrate recognizes the structure of the other, thereby altering its reactivity. Specifically, it was revealed that the positioning of one substrate within the enzyme pocket induces conformational changes in the other substrate, consequently influencing the nucleophilic reaction.