Triplet Quenching by Active Site Cysteine Residues Improves Photostability in Fatty Acid Photodecarboxylase
Ma, J.; Kalapothakis, J. M.; Spacey, H. J.; Johannissen, L. O.; Shrimpton-Phoenix, E.; Shanmugam, M.; Sakuma, M.; Wood, C. W.; Barran, P. E.; Heyes, D. J.; Scrutton, N. S.
Show abstract
Enzyme photobiocatalysis uses light to drive high-energy transformations but is limited by the rarity of photoenzymes. Fatty acid photodecarboxylase (FAP), a recently discovered photoenzyme, enables fatty acid conversion to alkanes/alkenes via excitation of an FAD cofactor, though its poor photostability and photoinactivation has hindered industrial applications. Here, we combine protein engineering approaches with biocatalytic and biophysical techniques, as well as computational chemistry, to demonstrate that additional active site cysteine residues can suppress oxygen-mediated inactivation processes that are driven by the FAD triplet-excited state. We identify a number of positions close to the FAD for cysteine residues that lead to a significant enhancement in activity as a result of an increase in the number of catalytic turnovers and improved photostability. The additional cysteine residues quench the triplet excited state of the FAD cofactor via a proposed proton-coupled electron transfer mechanism, resulting in lower levels of harmful reactive oxygen species. Our study highlights promising routes to mitigate non-productive, photoinactivation pathways in FAP and informs the rational design of new flavin-based photoenzymes.
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