UPOs see the light.
Nonspecific peroxygenases (UPOs) are enzymes that can selectively and efficiently transfer oxygen to organic compounds. UPO-catalyzed reactions are therefore suitable for the production of a wide variety of compounds because they can be used to activate carbon or heteroatoms that are inert or difficult to access. Since they are relatively stable, productive, and easy to handle, unlike other enzymes for oxofunctionalization, they are being intensively researched and optimized as potent tools for biocatalysis.
UPOs require only hydrogen peroxide as a co-substrate. However, at the same time, they suffer oxidative stress and thus an impairment of their activity from the hydrogen peroxide. Therefore, scientists are trying to develop methods to provide hydrogen peroxide to the reaction mixture only for catalysis, i.e. in a temporally and spatially defined way.
Scientists from IPB, MLU Halle, and the University of Regensburg recently presented a new, innovative method for hydrogen peroxide generation for UPO-catalyzed reactions. For this purpose, they made use of another type of enzyme, the flavin mononucleotide-containing fluorescent proteins (FbFP) from bacteria. These enzymes can act as photosensitizers, meaning when exposed to light, they convert molecular oxygen from the air into reactive oxygen species such as hydrogen peroxide with the help of the flavin cofactor. The production of the co-substrate for UPO can thus be switched on and off by light. While similar approaches using free flavin as a photosensitizer already existed, the Halle scientists were able to achieve significant improvements using FbFPs. First, they generated fusion proteins from an FbFP and a UPO, thereby bringing the light-driven generation of hydrogen peroxide into close proximity to the UPO, which consumes it as a co-substrate. Subsequently, the light-controllable fusion proteins were extensively optimized for expression in yeast using a previously established screening method (see news ticker #83). Second, genetically encoding the entire catalyst combination allows for flexible adjustments. Through targeted mutations and directed evolution, as well as the insertion of required promoters or linkers, constructs can be optimized for specific expression conditions, substrates, or conversions. Such an optimization via directed evolution had previously been realized for a UPO (see <link en public-relations press-releases press-detail newsticker-wissenschaft-95-biokatalysatoren>news ticker #95).
With the light-driven FbFP-UPO fusions, the scientists successfully achieved very efficient conversions, for example, the sulfoxidation of methyl phenyl sulfide or the stereoselective hydroxylation of indane to 1-(R)-indanol. Overall, the conversions could be doubled compared to the method using free flavin, and all this with a minimal reaction setup consisting of enzyme, substrate, buffer as electron source, and light as energy source for the reduction of atmospheric oxygen. The authors conclude that their new concept, which they call PhotUPO, opens up new possibilities in photo-biocatalysis and could be suitable for continuous-flow catalysis setups or in combination with other catalysis steps.
Original publication:
Light-Controlled Biocatalysis by Unspecific Peroxygenases with genetically-encoded Photosensitizers. Pascal Püllmann, Dominik Homann, Tobias A. Karl, Burkhard König, Martin J. Weissenborn. Angew. Chem. Int. Ed. 2023, 62, e202307897. https://doi.org/10.1002/anie.202307897