PFAS degrading enzymes and an Enzyme Classifier. The team contributed to science by identifying novel PFAS-degrading enzyme candidates, developing a chimera enzyme (A6T7 Chimera), designing 3 novel PFAS-degrading enzymes, and creating an expression classifier to predict protein expression in cell-free systems.
The classifier is helpful to scientists in the design of novel enzymes or proteins, it is not specific to any particular protein. Computationally generated proteins are really hard to express. This classifier can save us and others a lot of time. More specifically, an expression classifier will allow for pre-screening of all proteins before they go into the lab. Another reason why we are making an expression classifier is because we couldn't find any that work well. Our expression classifier will help future teams and scientists who choose to work with computationally generated proteins.
In our search to find specific dehalogenases proven to degrade PFAS, the team discovered the corrinoid iron-sulfur reductive dehalogenase A6RdhA. A6RdhA was identified within Acidimicrobium sp. Strain A6, a soil-dwelling microbe that, when incubated with a PFOA substrate, could partially defluorinate PFOA's structure (Fig. 1). PFOA is a legacy PFAS species that the EPA recently mandated in 2024 to be less than 4 parts per trillion (ppt), the minimal detectable limit, in drinking water.
When the gene coding for A6RdhA was knocked out, Acidimicrobium sp. Strain A6 lost its ability to degrade PFOA. This kind of concrete proof of PFAS degradation caught the team's attention, and we decided to focus our efforts towards reductive dehalogenases. Because A6RdhA has only recently been identified, its properties and complete structure have not been well characterized; in fact, the only known amino acid sequence of A6RdhA is missing a C-terminus of more than 100 amino acids. This incomplete fragment is predicted to be incapable of completing catalysis because the missing C-terminus makes up a large part of its active site. We posited that enzymes similar to A6RdhA would have similar PFAS degrading capabilities.
To find enzymes structurally similar to A6RdhA, we used the program Foldseek, which matches a query protein structure with proteins with similar structures within various protein structures and sequence databases. Through this method and a secondary literature review, the team identified 68 proteins with high structural similarity to A6RdhA (Fig. 2).
The majority of these enzymes had a relatively low annotation score on Uniprot and were not very well characterized structurally or functionally. However, many were classified as reductive dehalogenases, which aligns with the classification of A6RdhA and fits within the family of enzymes we were researching. In addition to having high structural similarity to a known PFAS degrading enzyme, the 68 were also sequentially diverse from each other.
At this point, the computational team formulated a separate plan that aimed to reconstruct the missing C-Terminus of A6RdhA, allowing us to produce a functional novel enzyme closer to A6RdhA in structure and, therefore, function than any other candidate. To achieve this goal, the team would produce a chimera enzyme made up of the fragment of A6RdhA and one of the structurally similar candidates. The candidate chosen to reconstruct A6RdhA was enzyme T7RdhA, a reductive dehalogenase with the second-highest structural similarity to A6RdhA out of the 68 candidates (Fig. 4). Another research group computationally verified T7RdhA to have PFAS degrading abilities similar to A6RdhA.
To construct the chimera, the structures of T7RdhA and the fragment of A6RdhA were aligned, and the point at which the fragment ends was identified. The point at which T7RdhA's sequence continues from the end of the fragment was identified. Every amino acid within T7RdhA's sequence from where the fragment sequence ends and T7RdhA's sequence begins was grafted onto the end of the A6RdhA fragment. This alteration of A6RdhA's structure reconstructed its active site.
While these projects took place, we also decided to select nine candidate enzymes to express and send to an off-site lab to be tested on a PFOA and GenX substrate. These nine candidates were chosen based on findings from our docking analysis and other factors. The complete list of candidates chosen and why they were chosen are shown in the table below. All expressed enzymes have been documented as Biobrick parts in the Registry.
