Engineering enzymes to expand substrate range.
Due to their high regio- or stereoselectivity, enzymes are very effective in selective oxygenations of compounds. Besides the predominant class of cytochrome P450-type enzymes, flavin-dependent monooxygenases are increasingly applied to hydroxylate organic substrate molecules. IPB researchers together with MLU colleagues have now performed a rational optimization of the prototypic flavin-dependent monooxygenase 4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) from E. coli to accept sterically more challenging substrates. Typically, 4HPA3H catalyzes the hydroxylation of a phenolic compound to its catechol form. The researchers investigated the crucial active site residues in 4HPA3H that lie nearby the substrate’s phenolic ring and compared them to those of related enzymes. Drawing from their observations, they exchanged few active site residues with rather large side chains for less bulky ones, thereby creating more space for substrates. While the wild-type enzyme accepts, e.g., p-coumaric acid and umbelliferone as substrates turning them into caffeic acid and esculetin, respectively, the altered enzyme was now able to accept and hydroxylate ferulic acid and naringenin – two larger substrates that could not be turned over by the wild-type form. Thus, the researchers show for the first time an engineered substrate expansion of a flavin-dependent monooxygenase enzyme for new, non-natural substrates, and with that, open up new possibilities for biotransformations.
Original publication:
Dippe, M., Herrmann, S., Pecher, P., Funke, E., Pietzsch, M. and Wessjohann, L. (2022), Engineered bacterial flavin-dependent monooxygenases for the regiospecific hydroxylation of polycyclic phenols. ChemBioChem. Accepted Author Manuscript. https://doi.org/10.1002/cbic.202100480
