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Structural insights from active site variants and β-8 loop interactions in viperin-like enzymes
Jake C Lachowicz1, Steven Grudman2, Jeffrey B Bonanno1
1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Structure (London, England : 1993)
|May 15, 2025
Summary
Viperin-like enzymes (VLEs) with a specific motif (NΦF) likely use CTP. Engineering the β-8 loop can switch VLE substrate selectivity, offering new ways to create alternative nucleoside triphosphates.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Viperin and viperin-like enzymes (VLEs) are radical SAM enzymes catalyzing dehydration of nucleoside triphosphates.
- VLEs exhibit species-dependent substrate selectivity, with some fungal VLEs possessing a distinct NΦF motif.
Purpose of the Study:
- To investigate the substrate specificity of NΦF VLEs.
- To understand the structural determinants of substrate selectivity in VLEs.
- To engineer VLEs for altered substrate preference.
Main Methods:
- Bioinformatics analysis
- Enzymatic assays
- X-ray crystallography
- Protein engineering
Main Results:
- NΦF VLEs were identified as likely CTP-utilizing enzymes.
- Engineering the β-8 loop of TvVip1 with a loop from a CTP-selective viperin successfully switched substrate selectivity from UTP to CTP.
- Structural insights into VLE substrate selectivity were gained.
Conclusions:
- The β-8 loop is a key determinant of substrate selectivity in VLEs.
- Engineering VLEs offers a potential strategy for producing alternative 3 ahydro-3 ahydro,4 ahydro-didehydronucleoside triphosphates (ddhNTPs).
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