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Engineered Biomolecular Recognition of RDX by Using a Thermostable Alcohol Dehydrogenase as a Protein Scaffold
Author(s) -
Bulutoglu Beyza,
Haghpanah Jennifer,
Campbell Elliot,
Banta Scott
Publication year - 2018
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201700539
Subject(s) - biopanning , pyrococcus furiosus , chemistry , alcohol dehydrogenase , combinatorial chemistry , biochemistry , protein engineering , computational biology , phage display , molecular recognition , peptide library , enzyme , biology , molecule , peptide , peptide sequence , organic chemistry , archaea , gene
There are many biotechnology applications that would benefit from simple, stable proteins with engineered biomolecular recognition. Here, we explored the hypothesis that a thermostable alcohol dehydrogenase (AdhD from Pyrococcus furiosus ) could be engineered to bind a small molecule instead of a cofactor or molecules involved in the catalytic transition state. We chose the explosive molecule 1,3,5‐trinitro‐1,3,5‐triazine (royal demolition explosive, RDX) as a proof‐of‐concept. Its low solubility in water was exploited for immobilization for biopanning by using ribosome display. Docking simulations were used to identify two potential binding sites in AdhD, and a randomized library focused on tyrosine or serine mutations was used to determine that RDX was binding in the substrate binding pocket of the enzyme. A fully randomized binding pocket library was selected, and affinity maturation by error‐prone PCR led to the identification of a mutant (EP‐16) that gained the ability to bind RDX with an affinity of (73±11) μ m . These results underscore the way in which thermostable enzymes can be useful scaffolds for expanding the biomolecular recognition toolbox.