Structural insights from active site variants and β-8 loop interactions in viperin-like enzymes

Viperin and viperin-like enzymes (VLEs) are members of the radical SAM superfamily that perform radical-mediated dehydrations on nucleoside triphosphates to yield 3′-deoxy-3′,4′-didehydronucleoside triphosphates (ddhNTPs). Interestingly, viperin and VLEs demonstrate species-dependent substrate selectivity. Some fungal species have a second VLE and, while most viperin and VLEs contain an NΦHX₄CX₃CX₂CF motif, these secondary VLEs are catalytically hindered by a histidine to phenylalanine substitution, an NΦFX₄CX₃CX₂CF motif (NΦF). Herein, we utilize a combination of bioinformatics, enzymology, and X-ray crystallography to demonstrate that NΦF VLEs likely utilize CTP as a substrate. Based on these observations, we demonstrate that the β-8 loop in TvVip1 can be engineered with the β-8 loop from a CTP-selective viperin (Mus musculus) to “swap” substrate selectivity from UTP to CTP. These results provide insight into the determinants of substrate selectivity exhibited by VLEs and introduce a potential route for engineering viperin and VLEs to form alternative ddhNTPs.