Production and purification of AtSOX
Aspergillus tubingensis strain D-85248 was obtained from the VTT Culture Collection  and cultivated as described in . A. tubingensis was grown on PeptoneTM-D(+)-glucose media adapted from [3, 5] in liquid cultivation at 30 °C under shaking (250 rpm). After removal of the fungal biomass by filtration, AtSOX was purified from the cell-free extract with four chromatographic steps according to the procedure described below. The culture supernatant was concentrated and buffer exchanged to 20 mM Tris-HCl pH 7 with a PD10 column (no 17-0851-01, GE Healthcare, Uppsala, Sweden), and applied on a QSepharose fast flow column (volume = 20 mL, Amersham Biosciences, Piscataway, USA). Proteins were eluted with a linear gradient from 0 to 0.3 M NaCl in 20 mM Tris-HCl pH 7. The SOX activity of the fractions was detected using 5,5-dithio-bis(2-nitrobenzoic acid) (Ellman’s reagent, DTNB, no D 218200, Sigma-Aldrich, Helsinki, Finland) and oxygen consumption measurements. The SOX active fractions were pooled and applied to a Superdex 75 column (volume = 24 mL, Amersham Biosciences, USA) using 50 mM Tris-HCl pH 7 buffer containing 150 mM NaCl at a 0.1 mL/min flow rate. SOX-active fractions were pooled, concentrated, buffer exchanged to 20 mM Tris-HCl pH 7, and applied twice to a Resource Q Sepharose column (volume = 1 mL, Amersham Biosciences, USA). In the first separation using Resource Q Sepharose resin, the proteins were eluted with a linear gradient from 0 to 0.2 M NaCl in 20 mM Tris-HCl pH 7, whereas in the second separation a shallower gradient from 0 to 0.11 M NaCl was used. Prepacked anion exchange columns were connected to ÄKTA™ chromatography system and UNICORN Control Software (GE Healthcare, Uppsala, Sweden).
Protein concentration was determined using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, USA) and bovine serum albumin (BSA, no. A8022, Sigma, St. Louis, USA) as a standard. Proteins were analysed by ready-made 12% Tris-HCl SDS-PAGE gel (no 161-1156, Bio-Rad, Hercules, USA) to ensure purity of the isolated AtSOX enzyme.
Amino acid analysis of AtSOX and strain identification
The partial amino acid sequence of AtSOX was determined from the N-terminus and internal peptide fragments. SDS-PAGE protein bands were stained with Coomassie Brilliant Blue, and the protein band of interest was excised and subjected to N-terminal sequencing. Edman degradation was performed using a Procise 494A protein sequencer from Perkin Elmer, Applied Biosystems Division (Foster City, CA, USA). The Coomassie stained protein band, assumably containing AtSOX, was cut off from the SDS-PAGE and in gel digested essentially as described by  for obtaining internal sequences and for peptide mass fingerprinting (PMF). In gel digestion was done by reducing proteins with dithiothreitol (DTT), alkylating with iodoacetamide and digesting with trypsin (no V5111, Promega, Madison, USA). The enzymatic cleavage occurred during overnight incubation at 37 °C. The peptides produced by enzymatic cleavage were analysed by MALDI-TOF MS after desalting using mC18 ZipTip (no ZTC18M096, Millipore, Billerica, USA). MALDI-TOF mass spectra of peptide fragments for PMF were obtained using an Ultraflex TOF/TOF instrument (Bruker-Daltonik GmbH, Bremen, Germany) using α-cyano-4-hydroxycinnamic acid (CHCA) as matrix. The sample solution was pipetted onto the sample plate together with matrix and air-dried. An electrospray ionization quadrupole time-of-flight tandem mass spectra for de novo sequencing were acquired using a Q-TOF instrument (Micromass, Manchester, UK) as described by . Protein identification with the generated data, the obtained peptide masses and the partial sequences, was performed using the Mascot Peptide Mass Fingerprint and MS/MS Ion Search programmes (http://www.matrixscience.com). Identification of the production strain (VTT D-85248) based on the morphology and DNA sequencing was performed at the Identification Services CBS (Utrecht, The Netherlands).
Peptide and protein substrate preparation
The reduced glutathione (GSH, no G4251), urinary trypsin inhibitor fragment (bikunin, no U-4751), and ribonuclease A (RNase A, no R5500) were purchased from Sigma (St. Louis, USA). Peptides, gliotoxin (no. A7665) and holomycin (no. sc-49,029), were purchased from PanReac AppliChem GmbH (Darmstadt, Germany) and Santa Cruz Biotechnology Inc. (Dallas, USA), respectively. The insulin B chain as Bunte salt derivative was kindly provided by Dr. Elisabeth Heine from Deutsches Wollforschungsinstitut (DWI, Aachen, Germany). Insulin chain B and RNaseA were reduced before use with 3% (v:v) 2-mercaptoethanol in the presence of 8 M urea according to the method used by . For the reduction, insulin B was dissolved in 50 mM (NH4)HCO3 pH 8.3 containing 8 M urea and 3% (v:v) 2-mercaptoethanol was used as solvent. In the case of RNaseA, 200 mM Tris-HCl buffer (pH 7.4) with urea and 2-mercaptoethanol was used. The solutions were incubated overnight at room temperature. The final concentration of insulin chain B was 1 mM, and concentration of RNaseA was 0.5 mM. After reduction the free sulfhydryl groups were detected with 5,5-dithio-bis(2-nitrobenzoic acid). Gliotoxin and holomycin were reduced with two equivalence of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) under nitrogen flow for 1 h prior liquid chromatography mass spectrometry (LC-MS) analysis.
Assay of AtSOX activity and pH and temperature behaviour
Different methods were used to measure AtSOX activity. First DTNB  was used for the detection of free sulfhydryl groups in the peptides after the enzymatic reaction. The spectroscopic measurements were done with a Cary 100 Bio UV-vis spectrophotometer (Varian Inc., Houten, the Netherlands). Second, the oxygen consumption measurement with Fibox 3 PreSens fiber-optic oxygen meter (Presens GmbH, Regensburg, Germany) was used to measure the changes in the concentration of dissolved oxygen during enzymatic reaction as described by . GSH (5 mM) was used as a substrate when determining AtSOX activity. The substrate was dissolved in phosphate buffered saline (PBS) containing 68 mM NaCl and 75 mM KH2PO4 pH 7.4. Activity of AtSOX was measured by the oxygen consumption assay in the presence of different GSH concentrations (0.25-10 mM) to determine the kinetic parameters. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) were determined with graphing software GraphPad Prism (GraphPad Software Inc., San Diego, USA) using nonlinear curve fitting to the Michaelis-Menten equation.
The thermal stability of AtSOX was determined at 30, 40, 50, 60 and 70 °C. The enzyme preparation was incubated in McIlvaine buffer pH 6.5 in a 0.2 mg/mL protein concentration for 1, 2, 15.5 and 20 h at 30 - 70 °C and also for 15 min at 70 °C, and the residual activity was measured by the oxygen consumption assay. pH stability was determined for AtSOX in a pH range between 2.3 and 10, and the residual enzyme activity was analysed by the oxygen consumption assay after 1 and 20 h incubation. pH optimum was determined by measuring AtSOX activity with the oxygen consumption assay using glutathione in McIlvaine citrate/phosphate buffer (pH 2.3-7.5), 50 mM Tris-HCl (pH 7-9) and 50 mM Glycine-NaOH (pH 8.5-10).
UV-vis absorption spectra were measured in 20 mM Tris-HCl pH 7.5 at 20 °C using a Cary 100/300 UV-vis spectrophotometer (Varian Inc., Houten, the Netherlands). In order to release a flavin cofactor, enzyme was thermally denaturated at 100 °C for 15 min followed by centrifugation (13,000 rpm, 10 min). Fluorescence was measured at 20 °C with a Cary Eclipse Fluorescence Spectrophotometer (Varian Inc., Houten, the Netherlands) using AtSOX solution in 20 mM Tris-HCl pH 7.5 in quartz cuvette with four optical faces. FAD fluorescence was recorded by exciting at 450 nm and monitoring emission between 450 and 600 nm.
Inhibition analysis of AtSOX
The AtSOX activity was analysed in a buffer solution and in the presence of different inhibitors. The effect of possible inhibitors on the AtSOX activity was determined using 5 mM GSH as substrate. The tested potential inhibitors were DTT, ethylenediaminetetraacetic acid (EDTA), potassium iodide, magnesium sulphate, manganese sulphate, sodium sulphate, sodium chloride, zinc sulphate, and sodium dodecyl sulphate (SDS). The inhibitors were tested at 10 mM concentration in 200 mM Tris-HCl buffer pH 7.5, and zinc sulphate was tested also at 1 mM concentration. The residual SOX activity was measured by oxygen consumption measurements (Fibox 3 PreSens fiber-optic oxygen meter, PreSens GmbH, Regensburg, Germany). Inhibition by zinc sulphate was confirmed by homovanillic acid (HVA) and peroxidase coupled assay as described in , and the assay was done according to  using GSH (5 mM) as a substrate. Chemicals, HVA (Cat. no. H1252) and peroxidase type II (Cat. no. P8250), were purchased from Sigma-Aldrich (St. Louis, USA). Fluorescence from the production of a HVA dimer was measured in a black 96-well microtiter plate at 320 nm excitation and 420 nm emission wavelengths using a Varioskan spectral scanning multimode microplate reader (Thermo Electron co., Vantaa, Finland).
Activity of AtSOX with reduced peptides
The ability of AtSOX to oxidise the peptides carrying free sulfhydryl groups was analysed by following the oxygen consumption. The activity of AtSOX (112 nkat) was analysed using reduced RNase A (0.25 mM solution in 200 mM Tris-HCl pH 7.4), and reduced GSH (5 mM solution in 75 mM KH2PO4 pH 7.4) as substrates in a total 1.86 mL reaction volume. The enzyme reactions were monitored by following the oxygen consumption of the co-substrate. The reactions were performed at room temperature and monitored for 5-20 min. For NMR spectroscopy, the enzymatically treated GSH was prepared by incubating the substrate with AtSOX for 20 min while the dissolved oxygen was totally consumed, as assessed by oxygen consumption measurements. Freshly prepared and 3 day old GSH solutions (5 mM) were used as a control samples to assess possible auto-oxidation. Reaction mixtures were analysed with 1H NMR as well as with 13C heteronuclear single quantum correlation (13C-HSQC) spectroscopy. The enzyme treated sample and the control sample were prepared in 75 mM potassium phosphate pH 7.4 in Shigemi NMR tubes. The NMR spectra were recorded on Varian INOVA 600 MHz NMR spectrometer at 293 K. One dimensional 1H spectra were recorded with water pre-saturation at 25 °C along with the gradient enhanced 13C-HSQC spectroscopy at 20 °C . Homonuclear Total Correlation Spectroscopy (TOCSY) experiments  were recorded to confirm the assignment.
Besides oxygen consumption method and NMR spectroscopy, the selected peptides were analysed with a MALDI-TOF MS on an Autoflex II spectrometer (Bruker Daltonik GmbH, Bremen, Germany) using CHCA matrix for peptides and sinapic acid (SA) for proteins. The reduced substrates GSH and insulin B were dissolved in 50 mM (NH4)HCO3, bikunin in 75 mM KH2PO4, and RNase A in 200 mM Tris-HCl pH 7.4. Purified SOX was used in the experiments in a dosage of 5.6 nkat (4.1 μg protein) and in case of insulin B and RNase A also in 56 nkat (41 μg protein). The enzymatic reactions were performed at 40 °C, except for bikunin that was incubated at room temperature for 20 h. Matrix solutions were prepared by dissolving CHCA or SA in a 1:1 solution of 0.1% trifluoroacetic acid and 100% acetonitrile. The spots for MALDI plate were prepared by using 1:1 proportion of matrix and sample. Typically 1 μl matrix and 1 μl sample were used for MALDI spot, where matrix was spotted first and dried before adding sample.
Reduced gliotoxin and holomycin were used in 200 μM final concentration in 0.1 M phosphate buffer (pH 6.5) for spectroscopic measurements. Gliotoxin and holomycin were used in 440 and 680 μM final concentrations, respectively, for LC-MS analysis. UV-vis absorption spectra from 200 to 600 nm were recorded with Varioskan spectral scanning multimode microplate reader (Thermo Electron co., Vantaa, Finland). The UV-vis absorption spectra from 200 to 600 nm was recorded for 10 min before and after addition of AtSOX (100 μl reaction volume). Reduced peptides incubated (50 min at ambient temperature) with AtSOX or with denaturated AtSOX were analysed with a ultra performance LC (UPLC) combined with a photodiode array detector and SYNAPT G2-S High Definition Mass Spectrometry (Waters, Milford Massachusetts, USA). One microliter of the sample was injected to a LC pre-column. LC-MS system was using a C18 Acquity UPLC VanGuard pre-column (2.1 × 5 mm, 1.7 μm,) and a C18 Acquity UPLC column (2.1 × 100 mm, 1.7 μm). All solvents used were spectral grade. Eluents were 5 mM ammonium acetate 0.1% formic acid in H2O (A) and in methanol (B). Elution was started with 10% B for 1 min, followed by a linear gradient from 10 to 100% B for 10 min and finally at 100% B for 2 min, with 0.4 mL min−1 flow. In these conditions reduced gliotoxin eluted from the LC column after 5.23 min and gliotoxin standard after 6.02 min. Elution times for reduced holomycin and holomycin standard were 1.69 and 3.74 min, respectively.
Analysis of SOX-coding genes in fungal genomes
The search was carried out as described in . In brief, scaffolds of 33 fungal genomes from  were divided in windows of 16 genes that overlapped with two genes. InterPro protein annotations of the genes were then used to look for windows that contained a flavin adenine dinucleotide (FAD)-dependent pyridine nucleotide-disulfide oxidoreductase (PNDR) that recognizes also SOX enzymes (InterPro: IPR000103), nonribosomal peptide synthetases (NRPS) (InterPro: IPR000873) or polyketide synthases (PKS) (InterPro: IPR001227) and cytochrome P450 monooxygenases (P450, InterPro: IPR001128) and/or Zn2Cys6 transcription factors (Zn2, InterPro: IPR001138). Searches were carried out and the results visualised with R essentially as described by .
A phylogenetic analysis of the selected 25 proteins from the protein family pyridine nucleotide-disulphide oxidoreductase, class-II (InterPro: IPR000103) was carried out. The selected protein sequences were obtained from UniProtKB except AtSOX sequence was retrieved from the genome of A. tubingensis from Joint Genome Institute (JGI) genome portal . The alignment of the sequences was done with MAFFT , and alignment was trimmed with trimAl . The aligned sequences were from Ascomycetes species except two proteins were bacterial origin, namely, DepH from Chromobacterium violaceum (UniProtKB: A4ZPY8) and HlmI from Streptomyces clavuligerus (UniProtKB: E2PZ87). A phylogenetic tree was constructed with FastTree  and visualised by Geneious version 10.0 created by Biomatters.