Trichothecenes are a class of mycotoxins that function as inhibitors of eukaryotic protein synthesis. Deoxynivalenol (DON), a.k.a. “vomitoxin”, is a trichothecene that contaminates crops, has been weaponized, and thus poses a health risk to livestock and humans. The epoxide moiety of trichothecenes is responsible for biological activity and de-epoxidation leads to detoxification. We propose to use directed evolution to re-engineer the active site of an epoxidase hydrolase to catalyze the epoxide ring opening reaction that will neutralize the DON mycotoxin. Directed evolution will be carried out using a fluorescence-based Bacterial Surface Display (BSD) system, recently developed in the Love Laboratory, in combination with the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor, or Cif. This enzyme was chosen because 1) it is a well-characterized epoxide hydrolase, 2) it is secreted by Pseudomonas aeruginosa and thus exhibits robust folding properties, and 3) its X-ray crystal structure has been solved to high resolution. Instead of redesigning Cif to hydrolyze DON in one step, we plan to re-engineer its active-site in a step-wise fashion using progressively larger synthetic epoxide substrates. To enhance this process we are currently engineering Cif to function as a Halo-Tag-type enzyme in which a stable covalent bond forms between an active-site Cif residue and the synthetic substrates. The Halo-Tag enzyme undergoes two-step catalysis in which a covalent intermediate forms between an active-site residue and the substrate. Mutation of a second-step residue results in stable bond formation between the enzyme and substrate. Cif undergoes a similar two-step mechanism and we are in the process of mutating active-site residues such that a stable, covalent intermediate forms between Cif and the progressively larger synthetic substrates. This will enhance the directed evolution efforts, as covalent capture will be used to select Cif variants from a library of mutants displayed using the BSD system.

Directed evolution of an epoxide hydrolase: Engineering the active site of the Cif epoxidase to form a covalent bond with progressively larger synthetic substrates

253rd National Meeting (2017)

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