Mechanism of diol dehydration by a promiscuous radical‐SAM enzyme homologue of the antiviral enzyme viperin (RSAD2)

3´‐deoxy nucleotides are an important class of drugs because they interfere with metabolism of nucleotides and their incorporation into DNA or RNA terminates cell division and viral replication. These compounds have largely been produced via multistep chemical synthesis and an enzyme with the ability to catalyse removal of 3´‐deoxy group from different nucleotides has yet to be described. Here, using a combination of HPLC, high‐resolution mass spectrometry, and NMR spectroscopy we demonstrate that a thermostable fungal radical S‐adenosylmethionine (SAM) enzyme with similarity to the vertebrate antiviral enzyme viperin (RSAD2) can catalyze transformation of CTP, UTP, and 5‐bromo‐UTP to their 3ʹ‐deoxy‐3′,4ʹ‐didehydro analogues. We show that unlike the fungal enzyme human viperin can only catalyse transformation of CTP. Using electron paramagnetic resonance (EPR) spectroscopy and molecular docking and dynamics simulations in combination with mutagenesis studies we provide insight into the origin of the unprecedented substrate promiscuity of the enzyme and the mechanism of dehydration of a nucleotide. Our findings highlight the evolution of substrate specificity in a member of the radical‐SAM enzymes. We predict that our work will help in utilizing a new class of radical‐SAM enzymes for biocatalytic synthesis of 3ʹ‐deoxy nucleotide/nucleoside analogues