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3'-Deoxynucleotides are an important class of drugs because they interfere with the metabolism of nucleotides, and their incorporation into DNA or RNA terminates cell division and viral replication. These compounds are generally produced by multi-step chemical synthesis, and an enzyme with the ability to catalyse the removal of the 3'-deoxy group from different nucleotides has yet to be described. Here, using a combination of HPLC, HRMS and NMR spectroscopy, we demonstrate that a thermostable fungal radical S-adenosylmethionine (SAM) enzyme, with similarity to the vertebrate antiviral enzyme viperin (RSAD2), can catalyse the transformation of CTP, UTP and 5-bromo-UTP to their 3'-deoxy-3',4'-didehydro (ddh) analogues. We show that, unlike the fungal enzyme, human viperin only catalyses the transformation of CTP to ddhCTP. Using electron paramagnetic resonance 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 using a new class of the radical-SAM enzymes for the biocatalytic synthesis of 3'-deoxy nucleotide/nucleoside analogues.

Original publication




Journal article



Publication Date





1605 - 1612


antiviral agents, nucleotide analogues, radical-SAM enzyme, tyrosyl radicals, viperin, Binding Sites, Biocatalysis, Crystallography, X-Ray, Cytidine Triphosphate, Fungal Proteins, Humans, Kinetics, Molecular Docking Simulation, Molecular Dynamics Simulation, Oxidoreductases Acting on CH-CH Group Donors, Phylogeny, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Proteins, Recombinant Proteins, S-Adenosylmethionine, Sordariales, Structural Homology, Protein, Substrate Specificity, Thermodynamics, Uridine Triphosphate