Crystallographic fragment screening-based study of a novel FAD-dependent oxidoreductase from Chaetomium thermophilum

The high-resolution crystal structure of a novel FAD-dependent oxidoreductase from the GMC oxidoreductase superfamily reveals a novel His–Ser active-site pair located in an extensive active-site pocket. Crystallographic fragment screening led to the identification of subsites inside the active-site pocket, indicating a preference for polyaromatic substrates.

DNA sequence encoding the CtFDO. The exons are highlighted in rose colour and these are spliced together into mRNA. For a recombinant production, the complete sequence.

Figure S2
Complete expected mature sequence of CtFDO with highlighted sequence parts confirmed with liquid chromatography-mass spectrometry (LC-MS/MS, yellow background) and with CtFDO crystal structures (grey dots). The sequence part with green background correspond to the signal peptide. Residues highlighted by cyan background mark the N-glycosylation sites in CtFDO. The red stars mark the only disulphide bridge. For the LC-MS/MS, the deglycosylated sample of CtFDO was digested by pepsin. Fragments were analysed by LC-MS/MS using an electrospray ion source of a 15T solariX XR FT-ICR mass spectrometer (Bruker Daltonics). Mass spectrometer was operated in positive, data dependent mode. Data were processed using the DataAnalysis 4.2 software and exported to mgf format. The fragments were identified by ProteinScape (Bruker Daltonics) with the Mascot search engine.

Figure S3
Mass distribution analysis done using mass photometry with (a) glycosylated and (b) deglycosylated form of CtFDO (CtFDOdegl). Graphs show a single peak for CtFDO and CtFDOdegl corresponding to 85 and 69 kDa containing more than 89 and 95 % of the population, respectively. No other oligomeric form was found. Data were collected on a Refeyn OneMP instrument using the DiscoverMP (v 2.2.1) software. Samples were diluted in 25 mM Tris-HCl with 75 mM NaCl, pH 7.5 to the concentration of 20 nM and 24 nM for CtFDO and CtFDOdegl, respectively. Data was analysed using DiscoverMP (version v. 2.3.dev12) using default settings.

Figure S4
MALDI-TOF spectra of CtFDO in (a) fully glycosylated and (b) deglycosylated form. One μl of sample at a concentration of 10 pmol•µl -1 was applied on stainless-steel MALDI target and let dry. The sample was overlaid with 1 µl of sinapinic acid (Sigma-Aldrich) and let dry at room temperature. Intact protein was analysed by a MALDI-TOF mass spectrometer Autoflex Speed (Bruker Daltonics) operated in linear positive mode. Data were processed by the FlexAnalysis 3.3 software (Bruker Daltonics).

Figure S5
Thermal stability of CtFDO fully glycosylated (black curve) and deglycosylated (CtFDOdegl) by endoglycosidase F1 (grey curve) measured by nano differential scanning fluorimetry using a Prometheus NT.48 instrument (NanoTemper). The samples of concentration approximately 0.7 mg•ml -1 were diluted in 25 mM Tris-HCl, pH 7.5 with 100 mM NaCl. The measurements were done in temperature range 20-95°C with slope 2.5 °C per min. Difference in the height of peaks is given by a slightly different concentration of the samples. The data were evaluated with the PR.ThermControl v2.1.1 software and plotted using GraphPad Prism 7.02 (GraphPad Software).

Figure S6
The UV-VIS absorption spectra for fully glycosylated (in black) and deglycosylated (in grey) CtFDO in solution showing two maxima at 390 nm and 460 nm. Both profiles show an oxidatized state of FAD in solution (25 mM Tris-HCl with 100 mM NaCl, pH 7.5). The UV-VIS absorption spectra were measured using a DeNovix DS-11 microvolume spectrophotometer with protein concentration of approximately 7.2 mg ml -1 . The different height of peaks is given by varied concentration of the samples. The spectra were plotted with GraphPad Prism version 7.02 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com).

Figure S7
The small-angle X-ray scattering (SAXS) plots obtained for deglycosylated form of CtFDO (CtFDOdegl). (a) The scattering plot (b) Guinier plot, and (c) dimensionless Kratky plot with s normalized by Rg (2.77 ± 0.08) and I(sRg) normalized to sRg = 0. The estimated molecular weight for CtFDOdegl ranges between 56 and 68 kDa depending on the calculation method (Hajizadeh et al., 2018) (59 kDa based on QP -protein volume from the Porod invariant, 68 kDa based on M0W, 56 kDa based on VCempirical Volume of Correlation). The SAXS experiment was performed in batch mode at the EMBL P12 beamline at the Petra III synchrotron radiation source (DESY, Hamburg) using a detector Pilatus 6M (Dectris, Baden-Daettwil, Switzerland). The measurement was done at 20 °C with sample-to-detector distance 3.0 m, wavelength 1.24 Å, and exposure time per image 0.045 s. The data were measured for CtFDOdegl (4.2 mg•ml -1 ), dissolved in 25 mM Tris-HCl, pH 7.5 with 25 mM NaCl. The data were processed using the ATSAS 3.0.0 package (Manalastas-Cantos et al., 2021). The data had fair quality according to the shape of the scattering curve, Guinier analysis and Kratky plot. Molecular envelope was not calculated.

Figure S8
Confirmation of CtFDO identity and presence of glycosylation site at Asn46 with MALDI-TOF peptide mass fingerprinting (PMF). The observed ion at m/z 2579.3108 corresponds to peptide N 42 VHSNYTFIISGGGISGLTLADR 64 modified by one N-acetyl-D-glucosamine unit linked to Asn46. PMF was performed using a sample of CtFDOdegl (amount of 4 μg) loaded onto SDS gel. SDS gel bands were cut-out, chopped into small pieces, and dehydrated by acetonitrile. Dithiothreitol at a concentration of 50 mM was added to the gel pieces and let incubate at 60 °C for 30 min. Then iodoacetamide at a concentration of 100 mM was added to the gel pieces. After 30 min in dark and at room temperature, the gel pieces were washed by water and dehydrated by acetonitrile. Trypsin solution was added to the gel pieces and let incubate at 37 °C overnight. One microliter of tryptic peptide mixture was applied on the stain-less steel MALDI target, covered with α-Cyano-4hydroxycinnamic acid as a matrix and analysed by a 15T solariX XR FT-ICR mass spectrometer (Bruker Daltonics) operating in positive mode. Data were processed by the DataAnalysis 4.2 software (Bruker Daltonics) and the mMass software.

Figure S9
Schematic diagram of the FAD interactions with CtFDO. Hydrogen bonds are indicated as red lines with donor-acceptor distances in Å (different values for chain B are given in parentheses). Besides the residues of CtFDO, the interaction with the active-site water (WAS) is indicated (red sphere).

Figure S10
The UV-VIS absorption spectra measured for CtFDOdegl crystal before (in black) and after (in grey) exposure to X-ray beam show the oxidized state and reduced state of FAD, respectively. The data prove reduction of FAD during exposure to X-rays. The absorption spectra were recorded with the OceanView spectroscopy software and normalized at 290 and 900 nm in GraphPad Prism version 7.02 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com).

Figure S11
UV-VIS absorption spectra of CtFDO reduction by dithionite (DTN) and re-oxidation by atmospheric molecular oxygen in solution. Reduced CtFDO lacks the flavin absorption peaks around 390 and 460 nm, typical for the oxidized state of FAD. The increased absorbance in the range of 300-360 nm corresponds to absorbance of DTN and its consumption in time. The solution was stirred by a pipette after 3 and 9 min. The re-oxidation effect was monitored with a UV/VIS spectrophotometer (Libra S22, Biochrom Ltd.) and the Resolution Spectrophotometer PC Software (Biochrom Ltd.). The spectra were buffer-subtracted. The spectra were plotted with GraphPad Prism version 7.02 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com).

Table S1
Summary of ligands and fragments utilized for preparation of crystals of CtFDO complexes using soaking or co-crystallization method.
For co-crystallization, the ligand concentration is given for reservoir solution and for soaking, it is given for crystallization drop. The concentration of fragments from Frag Xtal Screen is given as amount in crystallization drop, since most of them were not completely soluble in reservoir solution. marks solved structure of CtFDO:ligand complex.

Figure S19
The FAD pyrimidine moiety anchoring in CtFDO via hydrogen bonds with the main chain of the Ala133-Leu137 and Ser607-Ala610 peptides (carbon light grey) and the side chain of the "out" conformer of Ser607 (CtFDO:free, PDB entry: 6ZE2). The cofactor is shown with magenta carbons. The accomplishment of structural changes between the oxidized and reduced forms of the FAD isoalloxazine ring is not clear. One of the possibilities, movement of the dimethylbenzene moiety of FAD (accompanied by structural changes of the surrounding protein residues), is indicated by black arrow and theoretical conformation of planar isoalloxazine ring (carbon pink). The water molecule mediating hydrogen bonds between the FAD pyrimidine moiety and the peptides is shown as red sphere and is shown with all hydrophilic contacts. Hydrogen bonds of other atoms are indicated as dashed lines with labelled distances in Å (different values for chain B are given in parentheses). The graphics was created in Pymol Molecular Graphics System (Schrödinger).