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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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Fig. 1 Sequence similarity network at a bit-score cut-off of 420 for fungal sequences. The previously biochemically characterized sequences (details in Additional file 1: Table S1) and the sequences selected by this study (details in Additional file 1: Table S3) are shown with large symbols, while the remaining sequences are represented by grey dots. The sequences were colored based on specificity, and the shape reveals the characterization status (triangle: previously characterized; diamond: failed in production; square: successful production). The successfully produced enzymes with characterized activities are bolded and underlined, while the produced proteins without any detected activity are solely underlined. Enzyme function and naming is according to results in this study
Fig. 1 Sequence similarity network at a bit-score cut-off of 420 for fungal sequences. The previously biochemically characterized sequences (details in Additional file 1: Table S1) and the sequences selected by this study (details in Additional file 1: Table S3) are shown with large symbols, while the remaining sequences are represented by grey dots. The sequences were colored based on specificity, and the shape reveals the characterization status (triangle: previously characterized; diamond: failed in production; square: successful production). The successfully produced enzymes with characterized activities are bolded and underlined, while the produced proteins without any detected activity are solely underlined. Enzyme function and naming is according to results in this studyCC BY · PubMed Central
Fig. 2 The maximum likelihood trees of fungal AA3_2 sequences. a The maximum likelihood tree of all collected fungal AA3_2 sequences in this study. b, c Maximum likelihood trees of the AAO-PDH clade (b) and GOx-GDH clade (c). The phylogenetic clades that were defined by [4] are shown on the right side of the figure. The characterized proteins and the proteins that were produced successfully in this study are marked as solid circles. The proteins that were not successfully produced are marked as diamonds. The position of proteins within the AAO-PDH and GOx-GDH clades are indicated in Fig. 2b, c while the position of proteins outside the AAO-PDH and GOx-GDH clades are indicated in Fig. 2a. The sequences within the top 30 clusters in the SSN are highlighted in the branches
Fig. 2 The maximum likelihood trees of fungal AA3_2 sequences. a The maximum likelihood tree of all collected fungal AA3_2 sequences in this study. b, c Maximum likelihood trees of the AAO-PDH clade (b) and GOx-GDH clade (c). The phylogenetic clades that were defined by [4] are shown on the right side of the figure. The characterized proteins and the proteins that were produced successfully in this study are marked as solid circles. The proteins that were not successfully produced are marked as diamonds. The position of proteins within the AAO-PDH and GOx-GDH clades are indicated in Fig. 2b, c while the position of proteins outside the AAO-PDH and GOx-GDH clades are indicated in Fig. 2a. The sequences within the top 30 clusters in the SSN are highlighted in the branchesCC BY · PubMed Central
Fig. 3 Activity heat map of fungal AA3_2 proteins at pH 5 and 30 ℃. a The first-level screening on the substrate mixtures. b The second-level screening of individual substrates that were oxidized in the first-level mixture. Oxidase activity was assessed by coupling the H2O2 production to the oxidation of 2,2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) by horseradish peroxidase (HRP), while dehydrogenase activity was determined using BQ and DCIP as electron acceptors. The levels of the activity were defined based on the measured activity. High: > 1 U/mg; medium: 0.2–1 U/mg; moderate: 0.01–0.2 U/mg; and low: quantifiable—0.01 U/mg. Limited activity signified a minimum 10% difference in the absorbance between the reaction and the background after overnight incubation. (The activities of PcAA3_2A and ApAA3_2B’s were later proved to be false positives.)
Fig. 3 Activity heat map of fungal AA3_2 proteins at pH 5 and 30 ℃. a The first-level screening on the substrate mixtures. b The second-level screening of individual substrates that were oxidized in the first-level mixture. Oxidase activity was assessed by coupling the H2O2 production to the oxidation of 2,2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) by horseradish peroxidase (HRP), while dehydrogenase activity was determined using BQ and DCIP as electron acceptors. The levels of the activity were defined based on the measured activity. High: > 1 U/mg; medium: 0.2–1 U/mg; moderate: 0.01–0.2 U/mg; and low: quantifiable—0.01 U/mg. Limited activity signified a minimum 10% difference in the absorbance between the reaction and the background after overnight incubation. (The activities of PcAA3_2A and ApAA3_2B’s were later proved to be false positives.)CC BY · PubMed Central
Fig. 4 pH profiles of the four AA3_2s with high levels of activity. BQ was used as the electron acceptor. PsAaoA was assayed with p-anisyl alcohol, TaGdhA and ApGoxA were assayed with glucose, and KiOdhA was assayed with gentiobiose. Error bars represent the standard deviation of three replicated reactions
Fig. 4 pH profiles of the four AA3_2s with high levels of activity. BQ was used as the electron acceptor. PsAaoA was assayed with p-anisyl alcohol, TaGdhA and ApGoxA were assayed with glucose, and KiOdhA was assayed with gentiobiose. Error bars represent the standard deviation of three replicated reactionsCC BY · PubMed Central
Fig. 5 Conversion of aryl alcohols by AsAadhA and AsAadhB. The degree of oxidation was calculated from the product formation
Fig. 5 Conversion of aryl alcohols by AsAadhA and AsAadhB. The degree of oxidation was calculated from the product formationCC BY · PubMed Central
Fig. 6 The ability of each AA3_2 to use phenoxy radical as an electron acceptor. Reactions with a TaGdhA, b KiOdhA, and c ApGoxA were followed at 320 nm for the depletion of ferulic acid. d PsAaoA was followed at 350 nm for the formation of ferulic acid dimer. Reaction with laccase to KiOdhA in a ratio of 1–2 was also displayed in d for comparison. The enzymes were dosed according to the specific activity of laccase and AA3_2s. For the specific activity of TaGdhA and KiOdhA, BQ was used, and for the specific activity of ApGoxA and PsAaoA, oxygen was used
Fig. 6 The ability of each AA3_2 to use phenoxy radical as an electron acceptor. Reactions with a TaGdhA, b KiOdhA, and c ApGoxA were followed at 320 nm for the depletion of ferulic acid. d PsAaoA was followed at 350 nm for the formation of ferulic acid dimer. Reaction with laccase to KiOdhA in a ratio of 1–2 was also displayed in d for comparison. The enzymes were dosed according to the specific activity of laccase and AA3_2s. For the specific activity of TaGdhA and KiOdhA, BQ was used, and for the specific activity of ApGoxA and PsAaoA, oxygen was usedCC BY · PubMed Central

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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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