Peroxygenases and oxygenases within the tautomerase superfamily
|PhD ceremony:||Ms M. (Marie) Sigmund|
|When:||June 16, 2023|
|Supervisors:||prof. dr. G.J. (Gerrit) Poelarends, prof. dr. ir. M.W. (Marco) Fraaije|
|Where:||Academy building RUG|
|Faculty:||Science and Engineering|
Oxygenases and peroxygenases incorporate oxygen from the environmentally friendly oxidants O2 and H2O2, respectively, into their organic substrates in a stereo- and regioselective manner. While the majority of oxygenases and peroxygenases apply a cofactor, a few peculiar enzymes have been discovered, which are able to catalyse oxygenation and peroxygenation reactions without making use of a cofactor. However, the cofactor-independent oxygenases and peroxygenases known up to date are unsatisfactorily explored.
In the work reported in the thesis of Marie Sigmund, she investigated one tautomerase superfamily member with oxygenase activity, RhCC from Rhodococcus jostii, and one tautomerase superfamily member with promiscuous peroxygenase activity, 4 oxalocrotonate tautomerase (4 OT) from Pseudomonas putida.
Sigmund: "By screening selected enzymes homologous to RhCC, we discovered Rhop3 from Rhodococcus opacus with three-times enhanced oxygenase activity. However, the oxygenase activity of Rhop3 appears to be dependent on metal ions, disqualifying it as a cofactor-independent system.
Furthermore, we present an overview of enzymes with peroxygenase activity available in nature and discuss enzyme engineering strategies, which have been applied to improve their peroxygenase activity.
Applying directed evolution, we enhanced the promiscuous peroxygenase activity of a tandem-fused enzyme variant of 4 OT by about 150-times compared to 4-OT wild-type and realized the synthesis of α,β-epoxy-aldehydes in gram-scale with high enantioselectivity. Following, we applied this improved artificial peroxygenase in a one-pot multi-enzymatic cascade to synthesize enantioenriched epoxides and triols from biomass-derived starting materials."