Difference between revisions of "Part:BBa K5035003:Design"

Revision as of 04:15, 26 September 2024

A6RdhA was found in Acidimicrobium sp. strain A6, a microbe shown to degrade PFOA when incubated with a PFOA substrate. When the gene coding for A6RdhA was deactivated within A. sp. strain A6, the microbe lost the ability to degrade PFOA. These results suggested that A6RdhA can effectively cleave the strong carbon-fluorine bond present in all PFAS species. The complete amino acid sequence of A6RdhA was never fully recovered, leaving a fragmented sequence missing a C-terminus of over 100 amino acids. The known fragment of A6RdhA has an incomplete active site and, therefore, is predicted to be incapable of degrading PFOA. T7RdhA was computationally predicted to bind and interact with PFAS ligands and to have a high structural similarity to A6RdhA. The results from the analysis of T7RdhA's structure and function suggest it would have similar PFAS degradation abilities to A6RdhA.

When the structures of T7RdhA and the fragment of A6RdhA were aligned, 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, and when this chimera was computationally dimerized using Alphafold 3, its structure was predicted to be more stable than the initial fragment. When docked with PFOA and PFOS, the A6T7 chimera had high levels of predicted affinity to both. The PFOA and PFOS ligands were also predicted to have the most affinity within the reconstructed active site out of a set of predicted binding pockets, suggesting essential catalytic and structural features within the active site were now present.

The A6T7 Chimera has a hexahistidine tag for nickel column purification after expression.

https://static.igem.wiki/teams/5035/flowcharts-gels-graphs/igem-figure-4a.webp

References:

Guo, H.-B., Varaljay, V. A., Kedziora, G., Taylor, K., Farajollahi, S., Lombardo, N., Harper, E., Hung, C., Gross, M., Perminov, A., Dennis, P., Kelley-Loughnane, N., & Berry, R. (2023, March 11). Accurate prediction by AlphaFold2 for ligand binding in a reductive dehalogenase and implications for PFAS (per- and polyfluoroalkyl substance) biodegradation. Nature News. https://www.nature.com/articles/s41598-023-30310-x

Chiavola, A., Clément, J. C., Escapa, A., Huang, S., Lath, S., Lenka, S. P., Montagnolli, R. N., Ruiz-Urigüen, M., Sawayama, S., Senevirathna, S. T. M. L. D., Shuai, W., Sima, M. W., Yang, G., Zhang, D. Q., Chaudhuri, M. K., Cornell, R. M., Ding, L. J., & Gilson, E. R. (2024, February 15). Defluorination of pfas by Acidimicrobium sp. strain A6 and potential applications for remediation. Methods in Enzymology. https://www.sciencedirect.com/science/article/pii/S0076687924000168