Accelerating evolution
to break open a toxin

Published April 03 2017

Blight in barley crops caused by Fusarium graminearum IMAGE COURTESY OF COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION, AUSTRALIA

Vomitoxin makes animals — surprise — vomit. The fungal toxin also causes gastroenteritis, anorexia and immunosuppression. The compound is produced by the fungi Fusarium graminearum and Fusarium culmorum, which can colonize wheat and similar crops. While regulations by the U. S. Department of Agriculture prohibit its presence in food produced for human consumption at one part per million, a lack of similar restrictions for animal produce results in weight loss and death among pigs and cattle. Contamination normally leads to the destruction of infected crops. To come up with a solution, researchers at San Diego State University are using directed evolution to transform an enzyme found in Pseudomonas aeruginosa to degrade vomitoxin, which is formally known as deoxynivalenol.

Cif, shorthand for cystic fibrosis conductance regulatory inhibitory factor, is a virulence factor secreted by P. aeruginosa. The bacterium is especially dangerous when it colonizes the lungs of people with cystic fibrosis. As its name would suggest, the Cif enzyme plays a key role in this process.

The Cif enzyme is a member of the epoxide hydrolase class of enzymes that break open the carbon–oxygen epoxide ring of many small organic molecules by converting the oxygen bonds into two hydroxyl groups with the addition of a water molecule.

Vomitoxin’s structure contains an epoxide ring, which made the Cif enzyme an appealing choice for the researchers. “Our goal is to evolve this enzyme into a new enzyme that can bind and degrade the mycotoxin compound vomitoxin,” says John Love. Love is the primary investigator on the work that will be presented at the meeting by his graduate student, Myung Soo Ko.

“Another reason why we chose this enzyme is because its catalysis takes place in two steps,” says Love. “It’s a pretty standard mechanism.”

In the first step, the enzyme forms a covalent bond with the substrate. Amino acids at the enzyme’s active site then continue the catalysis and break bonds within the substrate, which, in this case, is the epoxide ring.

However, the Cif enzyme wasn’t made to bind to vomitoxin’s epoxide ring, so the researchers took to mutating its active site until it would.

In this process, “you randomize the DNA of the gene that codes for Cif,” says Love. “That creates diversity, so you have thousands, if not billions, of enzymes that are pretty much the same but have different amino enzymes in the active sites.”

That vast pool of mutated enzymes requires a filtering process, however, so the researchers developed a system known as bacterial surface display, or BSD. The system uses a combination of fused protein elements, anchoring transmembrane proteins and the fluorescent protein mCherry to drive the expression of a desired protein on the outer membrane of Escherichia coli. To express the Cif protein in this manner, the researchers cloned its gene into expression plasmids and transformed them into E. coli, where the protein was directed to the outer membrane by a leader sequence in the plasmid. Once the mutated enzymes were anchored there by the BSD protein elements, the researchers centrifuged the bacteria and conducted the catalytic analysis on the bacterial surface using the standard epoxide substrate epibromohydrin.

The researchers plan to evolve the enzyme to hydrolyze progressively larger epoxy substrates rather than seeking a mutation that immediately would cause the Cif enzyme to digest vomitoxin.

“Instead of hitting the home run, we’re hoping that we’re going to hit singles along the way,” says Love. “At the end of the project, we’ll have an enzyme that can successfully hydrolyze the epoxide deoxynivalenol.”

The researchers will present their results at the 2017 ASBMB Annual Meeting at the 12:30 p.m. poster session on April 25 in McCormick Place in Chicago.

John Arnst John Arnst is ASBMB Today’s science writer. Follow him on Twitter.