Reconstitution and substrate specificity for isopentenyl pyrophosphate of the antiviral radical SAM enzyme viperin.

Viperin is a radical SAM enzyme that has been shown to possess antiviral activity against a broad spectrum of viruses; however, its molecular mechanism is unknown. We report here that recombinant fungal and archaeal viperin enzymes catalyze the addition of the 5'-deoxyadenosyl radical (5'-dA•) to the double bond of isopentenyl pyrophosphate (IPP), producing a new compound we named adenylated isopentyl pyrophosphate (AIPP). The reaction is specific for IPP, as other pyrophosphate compounds involved in the mevalonate biosynthetic pathway did not react with 5'-dA• Enzymatic reactions employing IPP derivatives as substrates revealed that any chemical change in IPP diminishes its ability to be an effective substrate of fungal viperin. Mutational studies disclosed that the hydroxyl group on the side chain of Tyr-245 in fungal viperin is the likely source of hydrogen in the last step of the radical addition, providing mechanistic insight into the radical reaction catalyzed by fungal viperin. Structure-based molecular dynamics (MD) simulations of viperin interacting with IPP revealed a good fit of the isopentenyl motif of IPP to the active site cavity of viperin, unraveling the molecular basis of substrate specificity of viperin for IPP. Collectively, our findings indicate that IPP is an effective substrate of fungal and archaeal viperin enzymes and provide critical insights into the reaction mechanism.

Reconstitution and structure of a bacterial Pnkp1-Rnl-Hen1 RNA repair complex.

Ribotoxins cleave essential RNAs for cell killing, and RNA repair neutralizes the damage inflicted by ribotoxins for cell survival. Here we report a new bacterial RNA repair complex that performs RNA repair linked to immunity. This new RNA repair complex is a 270-kDa heterohexamer composed of three proteins-Pnkp1, Rnl and Hen1-that are required to repair ribotoxin-cleaved RNA in vitro. The crystal structure of the complex reveals the molecular architecture of the heterohexamer as two rhomboid-shaped ring structures of Pnkp1-Rnl-Hen1 heterotrimer fused at the Pnkp1 dimer interface. The four active sites required for RNA repair are located on the inner rim of each ring. The architecture and the locations of the active sites of the Pnkp1-Rnl-Hen1 heterohexamer suggest an ordered series of repair reactions at the broken RNA ends that confer immunity to recurrent damage.

Archaeal Elp3 catalyzes tRNA wobble uridine modification at C5 via a radical mechanism.

Approximately 25% of cytoplasmic tRNAs in eukaryotic organisms have the wobble uridine (U34) modified at C5 through a process that, according to genetic studies, is carried out by the eukaryotic Elongator complex. Here we show that a single archaeal protein, the homolog of the third subunit of the eukaryotic Elongator complex (Elp3), is able to catalyze the same reaction. The mechanism of action by Elp3 described here represents unprecedented chemistry performed on acetyl-CoA.

Molecular basis of bacterial protein Hen1 activating the ligase activity of bacterial protein Pnkp for RNA repair.

Ribotoxins cleave essential RNAs for cell killing in vivo, and the bacterial polynucleotide kinase-phosphatase (Pnkp)/hua enhancer 1 (Hen1) complex has been shown to repair ribotoxin-cleaved RNAs in vitro. Bacterial Pnkp/Hen1 is distinguished from other RNA repair systems by performing 3'-terminal 2'-O-methylation during RNA repair, which prevents the repaired RNA from repeated cleavage at the same site. To ensure the opportunity of 2'-O-methylation by bacterial Hen1 during RNA repair and, therefore, maintain the quality of the repaired RNA, Pnkp/Hen1 has evolved to require the participation of Hen1 in RNA ligation, because Pnkp alone is unable to carry out the reaction despite possessing all signature motifs of an RNA ligase. However, the precise role of Hen1 in RNA ligation is unknown. Here, we present the crystal structure of an active RNA ligase consisting of the C-terminal half of Pnkp (Pnkp-C) and the N-terminal half of Hen1 (Hen1-N) from Clostridium thermocellum. The structure reveals that the N-terminal domain of Clostridium thermocellum (Cth) Hen1, shaped like a left hand, grabs the flexible insertion module of CthPnkp and locks its conformation via further interaction with the C-terminal addition module of CthPnkp. Formation of the CthPnkp-C/Hen1-N heterodimer creates a ligation pocket with a width for two strands of RNA, depth for two nucleotides, and the adenosine monophosphate (AMP)-binding pocket at the bottom. The structure, combined with functional analyses, provides insight into the mechanism of how Hen1 activates the RNA ligase activity of Pnkp for RNA repair.