(309e) Modulating the Activity of Alcohol Dehydrogenase (AdhD) from Pyrococcus Furiosus via Applied Mechanical Forces

The effects of mechanical cues on biological processes have been well studied. However, most efforts have been concentrated on exploring the intrinsic properties of biomolecules by the exertion of forces on single molecules within isolated systems. In an enzymatic catalysis context, such isolated systems do not account for fundamental external factors such as intermolecular interactions and the microenvironment surrounding the enzyme. In this project, we focus on the modulation of the activity of a model enzyme, alcohol dehydrogenase D (AdhD), via applied mechanical forces under bulk catalysis conditions. In this project, we conjugate DNA springs on various binding sites on alcohol dehydrogenase D (AdhD) from Pyrococcus furious which is a member of the aldo-keto reductase (AKR) superfamily. The binding sites for the oligonucleotides are positioned on the enzyme across the active site and cofactor binding pocket to increase the likelihood of altering the key catalytic properties of the enzyme. In order to achieve this goal, dibenzocyclooctyne (DBCO) and amino-modified oligonucleotides are attached to the thiol and azido groups on the protein via addition and click chemistry reactions. These single-stranded DNA arms are then hybridized with a biotin-modified complementary strand to form a DNA spring surrounding the active site of the enzyme and separated using streptavidin-coated magnetic beads. The impact of the direction and magnitude of external mechanical forces on various enzymatic properties is explored through the utilization of different oligonucleotides and DNA attachment sites. So far, we have successfully shown an evident shift in the substrate specificity of an AdhD mutant which displays increased affinity for long chain secondary alcohols upon the attachment of a 50 base pairs long DNA spring. This result implies that the active site of the enzyme is being stretched due to the exertion of mechanical forces by the DNA spring as the wild type enzyme and unmodified mutant both have maximum affinity for shorter chain alcohols. The reversibility of this effect has also been shown by the introduction of a single cut to the spring via a restriction enzyme to stop the application of forces on the enzyme by the spring. As the use of applied mechanical forces in protein engineering is a new and underexplored area in protein engineering, our results exhibit the true potential this approach holds for reshaping enzymatic characteristics.