Enzymes are biocatalysts that accelerate practically all cellular reactions, i.e. the conversion of one substance (substrate) into another substance (product), and thus make life as we know it possible in the first place. Chemically, enzymes are proteins (protein molecules) and consist of a chain of amino acids, the sequence of which determines their spatial structure and thus also their respective function. The so-called "active centres" of enzymes are particularly important for their function. They consist of a few amino acids that bind the substrate and convert it into the product. Many substrates occur in two so-called enantiomeric forms, which are similar to each other. It is important to note that, as a rule, only one of the two enantiomers is bound and converted by the active centre, a property known as "enantioselectivity".
Due to their high efficiency, enzymes are also suitable for applications in industrial or medical environments. However, this requires their activities to be specifically modified and controlled. This is done by means of protein engineering, whereby one of the 20 naturally occurring amino acids is specifically exchanged for another. By reprogramming the cellular apparatus responsible for the production of proteins, it has recently become possible to replace natural amino acids with (unnatural) amino acids (UAS) that do not occur in cells. This opens up new possibilities for establishing and controlling enzyme activities. One particularly interesting option is the use of photosensitive UAS, which are incorporated near the active centre of enzymes and thus influence substrate binding and catalysis. In the work discussed here, this technique was applied to the enzyme phosphotriesterase (PTE), which enantioselectively degrades toxic substrates containing organophosphates to non-toxic products.
After incorporating the UAS at the active centre of the PTE, only one of the two enantiomers was initially bound. If the sample was then irradiated with ultraviolet light, thereby changing the chemical composition of the UAA, the other enantiomer could be bound instead and converted into the product. Using PTE as an example, the enantioselectivity of an enzyme could thus be artificially controlled by light for the first time. These data were explained by a subsequent detailed bioinformatic analysis based on the spatial structure of the PTE and the change of the active centre by the UAS. These results open up new possibilities for the precise spatio-temporal control of biochemical transformations with potential applications in the pharmaceutical-chemical industry.
The work was carried out in cooperation between the research groups of Prof Reinhard Sterner and Prof Till Rudack (Institute of Biophysics and Physical Biochemistry; Regensburg Center for Biochemistry) with Prof Frank Raushel (Department of Chemistry, Texas A & M University), who was at the University of Regensburg on a research sabbatical funded by a Humboldt Research Award.
Original publication
Caroline Hiefinger, Gabriel Zinner, Torben F. Fürtges, Tamari Narindoshvili, Sebastian Schindler, Astrid Bruckmann, Till Rudack, Frank M. Raushel, Reinhard Sterner (2025). Photo-Controlling the Enantioselectivity of a Phosphotriesterase via Incorporation of a Light-Responsive Unnatural Amino Acid. JACS Au 5, 858-870.
https://doi.org/10.1021/jacsau.4c01106 (external link, opens in a new window)
Foto: UR/Caroline Hiefinger