Identification and Characterization of PyrimidineNucleoside 2′-Hydroxylase

Functionalization of nucleosides at the 2′-position has become an important modification for therapeutic purposes to tailor pharmacological properties. The chemical synthesis of these molecules is challenging, and recent studies have explored bottom-up strategies with enzymes of the nucleoside salvage pathway. More than 50 years ago, a pyrimidine nucleoside 2′-hydroxylase (PDN2′H) activity had been described in fungal species extracts. However, the corresponding protein sequences were never reported and the protein characterization remained incomplete. This study describes the identification and characterization of PDN2′H from Neurospora crassa, which naturally hydroxylates thymidine at the α-2′-position as was now verified by NMR spectroscopy. Site-directed mutagenesis and biochemical assays indicated the protein to be an α-ketoglutarate-/Fe(II)-dependent dioxygenase. Furthermore, the substrate scope, phylogeny, and thermostability of <i>Nc</i>PDN2′H were determined and its enzymatic mechanism was elucidated by resolving its X-ray protein structure cocrystallized with thymidine. <i>Nc</i>PDN2′H is a long sought-after and important nucleoside-modifying addition to the biocatalytic portfolio.