Additional file 1 of Substrate specificity mapping of fungal CAZy AA3_2 oxidoreductases
Creators: name: Zhao, Hongbo
name: Karppi, Johanna
name: Mototsune, Owen
name: Poshina, Daria
name: Svartström, Jenny
name: Nguyen, Thi Truc Minh
name: Vo, Tri Minh
name: Tsang, Adrian
name: Master, Emma
name: Tenkanen, Maija
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Ecology
subject: Biological Sciences not elsewhere classified
subject: Plant Biology

description: Additional file 1: Table S1. List of the previously biochemically characterized proteins with the information of the database source, source organism, strain, protein activity, name, and the related publication. Table S2. Statistics of the major SSN clusters. Table S3. The AA3_2 sequences that were selected in this study with the database source, organism, and the production status and the biochemical information. Table S4. The list of substrates that were tested for the activity assays. Table S5. UPLC PDA retention time for each compound and the spectrum of each compound. Table S6. Substrate depletion by KiOdhA followed by HPAEC-PAD after 24 h incubation. Table S7. The extinction coefficient and wavelength to be used for the activity assay on aryl alcohols under different pH. Fig S1. SSN at the cut-off of 470 for the further division of cluster II. Fig S2. Absorption spectra of the concentrated AA3_2s. The oxidized FAD should have two absorbance maxima at 375-380 nm and at 440-444 nm. Fig S3. SDS page gel of the successfully produced AA3_2 proteins. Fig S4. UPLC-PDA Chromatogram (290 nm) of a) Standards of coniferyl alcohol, coniferaldehyde, ferulic acid, benzoquinone and hydroquinone b) Coniferyl alcohol after 8 h incubation with boiled PsAaoA at 30 °C (C) Coniferyl alcohol after PsAaoA oxidation for 8 h at 30 °C, showing the formation of coniferaldehyde. Fig S5. Mass spectra collected in negative ion mode showing a) Glucose b) Glucose after oxidation by ApGoxA and c) Glucose after oxidation by TaGdhA. Fig S6. Mass spectra collected in negative ion mode showing a) Gentiobiose b) Gentiobiose after oxidation by KiOdhA. Fig S7. Mass spectra collected in negative ion mode showing a) Glucose; b) Glucose after incubation with ApAA3_2B; c) Glucose after incubation with PcAA3_2A; and d) Glucose after incubation with McGdhA. Fig S8. Multiple Sequence Alignment (MSA) of characterized AA3_2 members in this study and previously. Red boxes show the primary sequence differences between the different enzymes. Fig S9. a) Surface and b) ribbon and sticks (active site and FAD) of the AlphaFold homology model of PsAaoA. The FAD and catalytic residues colored in green, hydrophobic residues to form the tunnel to block free access to active site are shown in orange, and the unique motifs identified from MSA are shown in Cyan. c) Alignment for the active site of PsAaoA (Red) and PeAAOx (white, PDB: 3FIM). Fig S10. a) Surface and b) ribbon and sticks (active site and FAD) of the AlphaFold homology model of TaGdhA. The FAD and catalytic residues colored in green, residues for substrate binding are shown in orange, and the unique motifs identified from MSA are shown in cyan. c) Alignment for the active site of TaGdhA (blue), AfGDH (white, PDB: 4YNT), and AfGDH in complex with D-glucono-1,5-lactone (pink, PDB: 4YNU). Fig S11. a) Surface and b) ribbon and sticks (active site and FAD) of the AlphaFold homology model of ApGoxA. The FAD and catalytic residues colored in green, residues for substrate binding are shown in orange, and the unique motifs identified from MSA are shown in cyan. c) Alignment for the active site of ApGoxA (blue), TcODH (white, PDB: 6XUT), and TcODH in complex with glucose (pink, PDB: 6XUU). Fig S12. a) Surface and b) ribbon and sticks (active site and FAD) of the AlphaFold homology model of AsAadhA. The FAD and catalytic residues colored in green and the unique motifs identified from MSA are shown in cyan. c) Alignment for the active site of AsAadhA (Red) and PeAAO (white, PDB: 3FIM). Fig S13. a) Surface and b) ribbon and sticks (active site and FAD) of the AlphaFold homology model of AsAadhB. The FAD and catalytic residues colored in green and the unique motifs identified from MSA are shown in cyan. c) Alignment for the active site of AsAadhB (Red) and PeAAO (white, PDB: 3FIM). Fig S14. a) Surface and b) ribbon and sticks (active site and FAD) of the AlphaFold homology model of KiOdhA. The FAD and catalytic residues colored in green, residues for substrate binding are shown in orange, and the unique motifs identified from MSA are shown in cyan. c) Alignment for the active site of KiOdhA (blue), TcODH (white, PDB: 6XUT), and TcODH in complex with glucose (pink, PDB: 6XUU). Fig S15. Amino acids and positions within the characterized AA3_2 sequences that are implicated in catalysis and substrate preference. Fig S16. Sequence logos of the active site residues from clades IIa, IIb, IV, VI, XI, XII, and XXX. The amino acid numbering of the sequences is based on PsAaoA for cluster IIa, AmPDH1 for cluster IIb, AsAadhA for cluster IV, TaGdhA for cluster VI, ApGoxA for cluster XI, AnGOx for cluster XII, and KiOdhA for cluster XXX.