Structural Insight into the Substrate Scope of Viperin and Viperin-like Enzymes from Three Domains of Life

Abstract

Viperin is a member of the radical S-adenosylmethionine superfamily and has been shown to restrict the replication of a wide range of RNA and DNA viruses. We recently demonstrated that human viperin (HsVip) catalyzes the conversion of CTP to 3′-deoxy-3′,4′-didehydro-CTP (ddhCTP or ddh-synthase), which acts as a chain terminator for virally encoded RNA-dependent RNA polymerases from several flaviviruses. Viperin homologues also exist in non-chordate eukaryotes (e.g., Cnidaria and Mollusca), numerous fungi, and members of the archaeal and eubacterial domains. Recently, it was reported that non-chordate and non-eukaryotic viperin-like homologues are also ddh-synthases and generate a diverse range of ddhNTPs, including the newly discovered ddhUTP and ddhGTP. Herein, we expand on the catalytic mechanism of mammalian, fungal, bacterial, and archaeal viperin-like enzymes with a combination of X-ray crystallography and enzymology. We demonstrate that, like mammalian viperins, these recently discovered viperin-like enzymes operate through the same mechanism and can be classified as ddh-synthases. Furthermore, we define the unique chemical and physical determinants supporting ddh-synthase activity and nucleotide selectivity, including the crystallographic characterization of a fungal viperin-like enzyme that utilizes UTP as a substrate and a cnidaria viperin-like enzyme that utilizes CTP as a substrate. Together, these results support the evolutionary conservation of the ddh-synthase activity and its broad phylogenetic role in innate antiviral immunity.

Chemoenzymatic Synthesis of 3′-Deoxy-3′,4′-didehydro-cytidine triphosphate (ddhCTP)

“…The viperin-like enzyme TtVip prefers UTP as a substrate but can also utilize CTP to produce ddhCTP. 22 Therefore, we cloned, expressed, and purified TtVip and set up a reaction to produce ddhCTP as previously described by Ebrahimi et al (see Supporting Information). 17 The reaction was incubated for ∼16 h, then applied to a 10 kDa MWCO spin concentrator to separate TtVip from the reaction products.…”

The vulnerability of radical SAM enzymes to oxidants and soft metals

“…Viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) is a human antiviral protein; it is also known as RSAD2 (radical S-adenosyl methionine domain containing 2) [ 26 , [41] , [42] , [43] ]. It can act upon CTP, converting it to an analogue that blocks viral replication, but the turnover number is low (0.2 min −1 ), leading some workers to propose that its authentic substrate may be something else [ 26 , 42 , 44 , 45 ]. Viperin homologs are present in many bacteria, archaea, and fungi.…”

Radical-SAM dependent nucleotide dehydratase (SAND), rectification of the names of an ancient iron-sulfur enzyme using NC-IUBMB recommendations

“…The fungal SAND has promiscuous activity and catalyses the dehydration of diverse nucleoside triphosphates (NTPs), e.g., CTP, UTP, and 5-bromo-UTP, to their ddh analogues via a mechanism requiring the transfer of an electron and a proton ( Figure 2B ) ( Ebrahimi et al, 2020b ). Next, a number of other groups characterised some microbial enzymes and showed that they catalyse dehydration of various NTPs to their ddh analogues ( Bernheim et al, 2021 ; Lachowicz et al, 2021 ) ( Figure 2C ). While the cellular function of these microbial proteins is not fully understood, the chemical reaction catalysed by SANDs can inhibit the activity of phage T7 RNA polymerase in E. coli ( Bernheim et al, 2021 ).…”