Mechanism of Diol Dehydration by a Promiscuous Radical‐SAM Enzyme Homologue of the Antiviral Enzyme Viperin (RSAD2)

• Published in: Chembiochem : a European journal of chemical biology 2020-06, Vol.21 (11), p.1605-1612
• Main Authors: Honarmand Ebrahimi, Kourosh, Rowbotham, Jack S., McCullagh, James, James, William S.
• Format: Article
• Language: English
• Subjects: Adenosylmethionine; Antiviral agents; Binding Sites; Biocatalysis; Cell division; Chemical synthesis; Crystallography, X-Ray; CTP; Cytidine triphosphate; Cytidine Triphosphate - chemistry; Cytidine Triphosphate - metabolism; Dehydration; Deoxyribonucleic acid; DNA; Electron paramagnetic resonance; Electron spin resonance; Enzymes; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Fungi; High-performance liquid chromatography; Homology; Humans; Kinetics; Liquid chromatography; Magnetic resonance spectroscopy; Metabolism; Molecular docking; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutagenesis; NMR; NMR spectroscopy; Nuclear magnetic resonance; Nucleoside analogs; Nucleotide analogs; nucleotide analogues; Nucleotides; Oxidoreductases Acting on CH-CH Group Donors; Phylogeny; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Proteins - chemistry; Proteins - genetics; Proteins - metabolism; radical-SAM enzyme; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Ribonucleic acid; RNA; S-Adenosylmethionine - chemistry; S-Adenosylmethionine - metabolism; Sordariales - chemistry; Sordariales - classification; Sordariales - enzymology; Spectroscopy; Spectrum analysis; Structural Homology, Protein; Substrate Specificity; Substrates; Thermodynamics; tyrosyl radicals; Uridine Triphosphate - analogs & derivatives; Uridine Triphosphate - chemistry; Uridine Triphosphate - metabolism; Vertebrates; viperin
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201900776

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. Fungal to antiviral: A new fungal radical‐SAM enzyme homologue of the human antiviral enzyme can catalyse the transformation of a range of natural or unnatural nucleotides to their analogues through a proton‐coupled electron transfer mechanism. The enzyme offers new biocatalytic routes for synthesis of 3′‐deoxy nucleotides/nucleosides as an important group of antiviral, antibacterial, or anticancer drugs.