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PreprintCited by 7Enzyme Production and CharacterizationEnzyme Catalysis and ImmobilizationEnzyme-mediated dye degradation

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Substrate specificity mapping of fungal CAZy AA3_2 oxidoreductases

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Zhao, Hongbo, Karppi, Johanna, Mototsune, Owen, Poshina, Daria, Svartström, Jenny, Nguyen, Thi Truc Minh, Vo, Tri Minh, Tsang, Adrian, Master, Emma, Tenkanen, Maija·2023·Biotechnology for Biofuels and Bioproducts
DOI 10.21203/rs.3.rs-3771693/v1PMC PMC10967070

Preprint — published as 10.1186/s13068-024-02491-8

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Abstract

Background Oxidative enzymes targeting lignocellulosic substrates are presently classified into various auxiliary activity (AA) families within the carbohydrate-active enzyme (CAZy) database. Among these, the fungal AA3 glucose–methanol–choline (GMC) oxidoreductases with varying auxiliary activities are attractive sustainable biocatalysts and important for biological function. CAZy AA3 enzymes are further subdivided into four subfamilies, with the large AA3_2 subfamily displaying diverse substrate specificities. However, limited numbers of enzymes in the AA3_2 subfamily are currently biochemically characterized, which limits the homology-based mining of new AA3_2 oxidoreductases. Importantly, novel enzyme activities may be discovered from the uncharacterized parts of this large subfamily. Results In this study, phylogenetic analyses employing a sequence similarity network (SSN) and maximum likelihood trees were used to cluster AA3_2 sequences. A total of 27 AA3_2 proteins representing different clusters were selected for recombinant production. Among them, seven new AA3_2 oxidoreductases were successfully produced, purified, and characterized. These enzymes included two glucose dehydrogenases (Ta GdhA and Mc GdhA), one glucose oxidase (Ap GoxA), one aryl alcohol oxidase (Ps AaoA), two aryl alcohol dehydrogenases (As AadhA and As AadhB), and one novel oligosaccharide (gentiobiose) dehydrogenase (Ki OdhA). Notably, two dehydrogenases (Ta GdhA and Ki OdhA) were found with the ability to utilize phenoxy radicals as an electron acceptor. Interestingly, phenoxy radicals were found to compete with molecular oxygen in aerobic environments when serving as an electron acceptor for two oxidases (Ap GoxA and Ps AaoA), which sheds light on their versatility. Furthermore, the molecular determinants governing their diverse enzymatic functions were discussed based on the AlphaFold structures. Conclusions The phylogenetic analyses and biochemical characterization of AA3_2s provide valuable guidance for future investigation of AA3_2 sequences and proteins. A clear correlation between enzymatic function and SSN clustering was observed. The discovery and biochemical characterization of these new AA3_2 oxidoreductases bring exciting prospects for biotechnological applications and broadens our understanding of their biological functions.

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Metadata

Authors
Zhao, H., Karppi, J., Mototsune, O., Poshina, D., Svartström, J., Nguyen, T. T. M., Vo, T. M., Tsang, A., Master, E., & Tenkanen, M.
Affiliations
  • University of Helsinki (FI)
  • Aalto University (FI)
  • University of Toronto (CA)
  • Concordia University (CA)
Corresponding author
Zhao, Hongbo
ORCID
Venue
Biotechnology for Biofuels and Bioproducts
Publisher
Research Square Platform LLC
Published
2023-12-21
Volume
17
Issue
1
Pages
47-47
ISSN
2731-3654 (ISSN-L)
DOI
10.21203/rs.3.rs-3771693/v1
PMC
PMC10967070
OpenAlex
W4393233240
Cited by
7
References
1
Citations by year
2026: 2 · 2025: 5
FWCI
1.31
Type
journal article
Language
en
Open access
Licence
Locations
  • Biotechnology for Biofuels and Bioproducts (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Aaltodoc (Aalto University) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Työväentutkimus Vuosikirja (submittedVersion)
Funders
  • Helsingin Yliopisto
  • Helsingin ja Uudenmaan Sairaanhoitopiiri
  • Academy of Finland (308996)
  • Suomen Kulttuurirahasto (00211192)
  • Novo Nordisk
  • Novo Nordisk Fonden (0070816)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Enzyme-mediated dye degradation
Keywords
carbohydrate-active enzymes, fungal enzymes, substrate specificity, glucose dehydrogenase, glucose oxidase, aryl-alcohol oxidase, enzyme function prediction, homology modeling

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