Discovery of a fungal copper radical oxidase with high catalytic efficiency towards 5-hydroxymethylfurfural and benzyl alcohols for bioprocessing

Mathieu, Yann; Offen, Wendy A.; Forget, Stephanie M.; Ciano, Luisa; Viborg, Alexander Holm; Blagova, Elena; Henrissat, Bernard; Walton, Paul H; Davies, Gideon J.; Brumer, Harry

Abstract

Copyright © 2020 American Chemical Society. Alternatives to petroleum-based chemicals are highly sought-after for ongoing efforts to reduce the damaging effects of human activity on the environment. Copper radical oxidases from Auxiliary Activity Family 5/Subfamily 2 (AA5_2) are attractive biocatalysts because they oxidize primary alcohols in a chemoselective manner without complex organic cofactors. However, despite numerous studies on canonical galactose oxidases (GalOx, EC 1.1.3.9) and engineered variants, and the recent discovery of a Colletotrichum graminicola copper radical alcohol oxidase (AlcOx, EC 1.1.3.13), the catalytic potentials of very few AA5_2 members have been characterized. Guided by the sequence similarity network and phylogenetic analyses, we targeted a distinct paralog from the fungus C. graminicola as a representative member of a large uncharacterized subgroup of AA5_2. Through recombinant production and detailed kinetic analysis, we demonstrated that this enzyme is weakly active toward carbohydrates but efficiently catalyzes the oxidation of aryl alcohols to the corresponding aldehydes. As such, this represents the initial characterization of a demonstrable aryl alcohol oxidase (AAO, EC 1.1.3.7) in AA5, an activity which is classically associated with flavin-dependent glucose-methanol-choline (GMC) oxidoreductases of Auxiliary Activity Family 3 (AA3). X-ray crystallography revealed a distinct multidomain architecture comprising an N-terminal PAN domain abutting a canonical AA5 seven-bladed propeller catalytic domain. Of direct relevance to biomass processing, the wild-type enzyme exhibits the highest activity on the primary alcohol of 5-hydroxymethylfurfural (HMF), a product of significant interest in the lignocellulosic biorefinery concept. Thus, the chemoselective oxidation of HMF to 2,5-diformylfuran (DFF) by C. graminicola aryl alcohol oxidase (CgrAAO) from AA5 provides a fundamental building block for chemistry via biotechnology.

Citation

Mathieu, Y., Offen, W. A., Forget, S. M., Ciano, L., Viborg, A. H., Blagova, E., Henrissat, B., Walton, P. H., Davies, G. J., & Brumer, H. (2020). Discovery of a fungal copper radical oxidase with high catalytic efficiency towards 5-hydroxymethylfurfural and benzyl alcohols for bioprocessing. ACS Catalysis, 10(5), 3042-3058. https://doi.org/10.1021/acscatal.9b04727

• Journal Article Type: Article
• Acceptance Date: Feb 3, 2020
• Online Publication Date: Feb 3, 2020
• Publication Date: Mar 6, 2020
• Deposit Date: Feb 17, 2020
• Publicly Available Date: Feb 4, 2021
• Journal: ACS Catalysis
• Electronic ISSN: 2155-5435
• Publisher: American Chemical Society
• Peer Reviewed: Peer Reviewed
• Volume: 10
• Issue: 5
• Pages: 3042-3058
• DOI: https://doi.org/10.1021/acscatal.9b04727
• Keywords: oxidoreductases, enzyme kinetics, structural biology, biocatalysis, bioproducts, EC 1.1.3.7, EC 1.1.3.47
• Public URL: https://nottingham-repository.worktribe.com/output/3978130
• Publisher URL: https://pubs.acs.org/doi/10.1021/acscatal.9b04727#
• Additional Information: This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Catalysis, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://pubs.acs.org/doi/10.1021/acscatal.9b04727#