Part:BBa_K5035007:Design

O68252 is a Tetrachloroethene reductive dehalogenase produced by Sulfurospirillum multivorans (Dehalospirillum multivorans). O68252 catalyzes the reductive dechlorination of tetrachloroethene (PCE) to trichloroethene (TCE) and of trichloroethene to cis-1,2-dichloroethene (DCE). O68252 is structurally similar to enzyme A6RdhA, a corrinoid iron-sulfur-containing reductive dehalogenase capable of degrading PFOA. Similar to A6RdhA, O68252 requires iron, sulfur, and norpseudovitamin B12 as cofactors. Because of O68252's structural and familial similarity to A6RdhA, it is predicted to have similar PFOA degrading abilities.

O68252 requires sulfur, iron, and norpseudo vitamin B12 to fold properly. The 2024 IEA iGEM team successfully expressed O68252 within MyTXTL and incorporated the necessary cofactors within its structure using the cofactor reconstitution protocol detailed by Nakamura et al. The Results of the team's expression of O68252 are shown in the gel below.

Protein gel electrophoresis of purified O68252 solution shows a band at the expected size of around 46 kDa.

Design Notes

A hexahistidine tag for nickel column purification was added to O68252.

References

https://www.uniprot.org/uniprotkb/O68252/entry

Nakamura, R., Obata, T., Nojima, R., Hashimoto, Y., Noguchi, K., Ogawa, T., & Yohda, M. (2018). Functional Expression and Characterization of Tetrachloroethene Dehalogenase From Geobacter sp. Frontiers in microbiology, 9, 1774. https://doi.org/10.3389/fmicb.2018.01774