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Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase

preprint
submitted on 24.11.2020, 08:09 and posted on 24.11.2020, 13:10 by Anja Knorrscheidt, Jordi Soler, Nicole Hünecke, Pascal Püllmann, Marc Garcia-Borràs, Martin Weissenborn
Unspecific peroxygenases (UPOs) enable oxyfunctionalisations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed a protein engineering in S. cerevisiae with the novel MthUPO. Experimental approaches were combined with computational modelling resulting in the screening of more than 5,300 transformants. This protein engineering led to a significant reshaping of the active site as elucidated by molecular dynamics. The kcat/Km was improved by 16.5-fold. Variants were identified with high chemo- and regioselectivities in the oxyfunctionalisation of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with excellent enantioselectivities of 95 % ee. Additionally, the first reported effective exchange of the conserved catalytic Glu residue was observed.

History

Email Address of Submitting Author

martin.weissenborn@ipb-halle.de

Institution

Leibniz-Institut für Pflanzenbiochemie & MLU Halle Wittenberg

Country

Germany

ORCID For Submitting Author

0000-0002-1200-4485

Declaration of Conflict of Interest

None

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