Enantioselective High‐Throughput Assay Showcased for the Identification of (R)‐ as well as (S)‐Selective Unspecific Peroxygenases for C−H Oxidation

Author:

Swoboda Alexander1,Pfeifenberger Lukas Johannes12,Duhović Zerina1,Bürgler Moritz2,Oroz‐Guinea Isabel3ORCID,Bangert Klara3,Weißensteiner Florian3,Parigger Lena12,Ebner Katharina2,Glieder Anton2,Kroutil Wolfgang1345ORCID

Affiliation:

1. Austrian Center of Industrial Biotechnology (ACIB GmbH) c/o Department of Chemistry University of Graz Heinrichstraße 28 8010 Graz Austria

2. Bisy GmbH Wünschendorf 292 8200 Hofstätten an der Raab Austria

3. Department of Chemistry University of Graz Heinrichstraße 28 8010 Graz Austria

4. BioTechMed Graz 8010 Graz Austria

5. Field of Excellence BioHealth University of Graz 8010 Graz Austria

Abstract

AbstractIdentifying (bio)catalysts displaying high enantio‐/stereoselectivity is a fundamental prerequisite for the advancement of asymmetric catalysis. Herein, a high‐throughput, stereoselective screening assay is reported that gives information on enantioselectivity, stereopreference and activity as showcased for peroxygenase‐catalyzed hydroxylation. The assay is based on spectrophotometric analysis of the simultaneous formation of NAD(P)H from the alcohol dehydrogenase catalyzed enantioselective oxidation of the sec‐alcohol product formed in the peroxygenase reaction. The assay was applied to investigate a library comprising 44 unspecific peroxygenases (UPOs) containing 25 UPOs not reported yet. Thereby, previously non‐described wild‐type UPOs displaying (S)‐ as well as (R)‐stereoselectivity for the hydroxylation of representative model substrates were identified, reaching up to 98 % ee for the (R)‐ and 94 % ee for the (S)‐enantiomer. Homology models with concomitant docking studies indicated the structural reason for the observed complementary stereopreference.

Funder

Steirische Wirtschaftsförderungsgesellschaft

Österreichische Forschungsförderungsgesellschaft

Karl-Franzens-Universität Graz

HORIZON EUROPE Marie Sklodowska-Curie Actions

Publisher

Wiley

Subject

General Medicine

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