Identification and Characterisation of Pyrimidine Nucleoside 2'-Hydroxylase

Functionalisation of nucleosides at the 2'-position has become an important modification for therapeutic purposes to tailor pharmacological properties. Chemical synthesis of these molecules is challenging, and recent studies have explored bottom-up strategies with enzymes of the nucleoside salvage pathway and late-stage functionalisation capabilities. More than 55 years ago, a pyrimidine nucleoside 2'-hydroxylase (PDN2'H) activity was described in three fungal species. However, the corresponding protein sequences have never been reported. This study describes the identification and characterisation of PDN2'H from Neurospora crassa, which naturally hydroxylates thymidine at the 2'-position as now verified by NMR. Site-directed mutagenesis confirmed the protein to be an α-ketoglutarate-/Fe(II)-dependent dioxygenase. We performed investigation of its substrate scope, phylogeny, thermostability and elucidated the enzymatic mechanism with help of PDN2'H’s crystal structure co-crystallised with thymidine. This work adds a long sought-after and important nucleoside-modifying protein to the biocatalytic portfolio.