Engineered Biomolecular Recognition of RDX by Using a Thermostable Alcohol Dehydrogenase as a Protein Scaffold.

Abstract

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) μm. These results underscore the way in which thermostable enzymes can be useful scaffolds for expanding the biomolecular recognition toolbox.