Plasmid construction, protein expression, purification and in vitro assays
To construct plasmids for protein expression of EasC from C. fusiformis (EasCCf; GenBank no. ABV57821.1) and A. fumigatus (EasCAf; GenBank accession no. XM_751047.1), the DNA fragments were amplified using the corresponding primers and inserted into NdeI- and XhoI-digested pET28a using the Gibson assembly method. The plasmids for enzyme mutants were constructed by amplifying the wild-type template using corresponding primers by means of a fast mutagenesis system kit. The primers used are listed in Supplementary Table 1. The plasmids were transformed into Escherichia coli BL21(DE3) competent cells for protein expression. Precultures were grown overnight in Luria–Bertani (LB) medium (10 ml) containing 50 µg ml−1 of kanamycin at 37 °C with shaking at 250 rpm. Then, 10 ml of preculture was transferred to 1 l of fresh LB medium with 50 µg ml−1 of kanamycin and further incubated at 37 °C with shaking at 250 rpm. When the optical density at 600 nm reached 0.6–0.8, the expression of EasC was induced by 5 µM isopropyl β-d-thiogalactopyranoside supplemented with 10 µM chlorhematin (chlorohemin or chloroprotoporphyrin IX iron(III)) and cultured for 18 h at 16 °C and 200 rpm. Cells were collected by centrifugation at 5,000 rpm for 30 min and suspended in buffer A containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl and 20 mM imidazole, followed by disruption using a French press (JuNeng Biology & Technology). The cell lysate was centrifuged at 4 °C for 50 min at 17,000g to remove the cell debris. The supernatant was applied to a fast protein liquid chromatography system (GE HealthCare) coupled with a Ni–NTA column and eluted with a gradient of 20–500 mM imidazole in buffer A. Purified proteins were dialysed and concentrated to 10 mg ml−1 in buffer A without imidazole. The SDS–PAGE analyses of the wild-type and variant EasCCf proteins are shown in Supplementary Fig. 18.
A typical 50 μl of the assay solution containing 50 mM Tris-HCl buffer (pH 7.5), 40–100 μM enzymes, 2 mM NADPH and 0.5 mM substrate was prepared for testing the enzymatic activities of EasC. The reactions were generally performed at 30 °C for 1 h and quenched by adding 100 μl of methanol. Protein precipitate from the reactions was removed by centrifugation. The supernatant was then analysed using HPLC and liquid chromatography–mass spectrometry (LC–MS). The enzyme activity of each mutant was calculated as a percentage of wild-type enzyme, which was set at 100%.
Protein purification, cryo-EM data acquisition and processing, model building and refinement of EasCCf
To solve the protein structure, the protein solution of EasCCf obtained by performing the aforementioned procedure was further purified through size exclusion chromatography coupled with a Superdex 200 10/300 GL column (GE HealthCare). Peak fractions were collected for cryo-EM sample preparation. For complex structure, the recombinant EasCCf protein was incubated with 10 mM PCC for 30 min before cryo-sample preparation.
To prepare cryo-EM samples, 4 μl of purified EasCCf (0.5 mg ml−1) or substrate-engaged EasCCf (0.5 mg ml−1) was applied onto glow-discharged holey carbon grids (Quantifoil Cu R1.2/1.3; 300 mesh). The grids were then blotted for 5 s at 4 °C with 100% humidity and plunge frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher Scientific). High-quality grids were loaded onto a Titan Krios electron microscope, operated at 300 kV, for data collection. Image stacks were recorded with a K3 detector (Gatan) using EPU (Thermo Fisher Scientific) in super-resolution counting mode at a nominal magnification of 105,000× corresponding to a calibrated pixel size of 0.425 Å. Each stack of 40 frames was exposed for 2.5 s with preset defocus values ranging from −1.0 to −2.4 μm, and the total dose for each stack was approximately 52 e−/Å2.
All cryo-EM data processing was performed using CryoSPARC v.3.3.1 (ref. 47). In general, the video stacks were motion-corrected using patch motion correction, and the micrographs were binned to 0.85 Å per pixel. These images were used for contrast transfer function estimation using a patch-based contrast transfer function. For apo-form EasCCf, a total of 1,002 good micrographs were selected, and 590,067 particles were automatically picked using the CryoSPARC Blob Picker and extracted with a box size of 256 pixels. After two rounds of two-dimensional classification, 296,958 particles were selected, followed by one round of ab initio reconstruction. The dataset was processed with C1 and C2 symmetries, which yielded similar maps and models. Thus, C2 symmetry was applied in subsequent analyses. Particles were subjected to one round of heterogeneous refinement on the basis of two better classes to further remove bad particles. After one round of non-uniform and local refinement, a final dataset of 184,130 particles was reconstructed to produce a 2.64 Å resolution map (Supplementary Fig. 3). The substrate-engaged EasCCf data were processed using the same workflow. A total of 2,027 good micrographs were selected, and 1,155,245 particles were picked automatically. After two rounds of two-dimensional classification, 839,745 particles were selected for one round of ab initio reconstruction. Similar to the case for the apo-form EasCCf, maps were generated using C1 and C2 symmetries. C2 symmetry was applied to produce the final maps. After one round of heterogeneous refinement and one round of non-uniform and local refinement, a final dataset of 603,385 particles was reconstructed to produce a 2.33 Å resolution map (Supplementary Fig. 4). All maps were subjected to a sharpening process using the Sharpening Tools in CryoSPARC, and the resolution of the final maps was estimated by gold-standard Fourier shell correlation using the 0.143 criterion.
The atomic model of apo-form EasCCf built using AlphaFold2 (ref. 48) was docked into the apo-form EasCCf map in UCSF Chimera49. The model was manually adjusted in Coot v.0.9.6 (ref. 50), followed by refinement against the corresponding map using the phenix.real_space_refine program in Phenix v.1.20 with geometry and secondary structure restraints imposed51. The model of substrate-engaged EasCCf was built starting with the model of the apo-form EasCCf. All final models were evaluated using MolProbity52. The statistics of map reconstruction and model building are summarized in Supplementary Table 2.
HPLC and LC–MS analysis
The conversion of biochemical reactions was calculated according to the relative peak area of the substrate analysed using HPLC or LC–MS at a UV detection wavelength of 320 nm (for PCC).
For method A, samples were analysed by Waters 2596 equipped with a mass spectrum detector (2996) and a C18 column (Agilent Eclipse XDB-C18; 2.1 × 100 mm; 3.5 μm) and eluted with a gradient method (0–100% mobile phase B in 15 min, with H2O and MeCN as mobile phases A and B, respectively) using a flow rate of 0.3 ml min−1. For method B, samples were analysed by CORUI HPLC system equipped with a C18 analytic column (Agilent Eclipse Plus C18; 4.6 × 250 mm; 5.0 μm) and eluted with a gradient method (0–100% mobile phase B in 20 min, with H2O and MeCN as mobile phases A and B, respectively) using a flow rate of 1.0 ml min−1.
Molecular docking
Molecular docking was performed using the CDOCKER procedure in Discovery Studio Client v.19.1.0.
NaN3 and NH2OH inhibitory assay
To test the inhibitory effect of NaN3 and NH2OH on EasCCf, we prepared a 50-μl reaction solution consisting of 50 mM Tris-HCl buffer (pH 7.5), 40 μM enzymes, 0.5 mM substrate, 2 mM NADPH and several concentrations of sodium azide (320→0.039 mM) and hydroxylamine (25→0.006 mM). The reactions were incubated for 1 h at 30 °C and quenched with 100-μl methanol. Proteins in the reactions were removed by centrifugation. The supernatant was then analysed using LC–MS. Inhibition potency (IC50) values were calculated using a four-parameter logistic equation using GraphPad Prism, v.6.0 (GraphPad Software). Percentage of inhibition = (substrate residual rate with inhibitors − substrate residual rate without inhibitors) × 100%.
ROS inhibitory assay
To evaluate the ROS inhibitory effect, we prepared a 50-μl reaction solution that included 50 mM Tris-HCl buffer (pH 7.5), 40 μM enzymes, 0.5 mM substrate, 2 mM NADPH and different concentrations of scavengers. The reactions were implemented for 1 h at 30 °C and then quenched with 100 μl methanol. Proteins in the reactions were removed by centrifugation. The supernatant was then analysed by LC–MS. The enzyme activity of each sample was calculated as a relative value, with the activity in the absence of inhibitors set at 100%
Superoxide generation assay for EasC activity restoration
For in vitro studies, we prepared a 50-μl reaction solution containing 50 mM Tris-HCl buffer (pH 7.5), 20 μM enzymes, 0.5 mM substrate, 2 mM NADPH, 10 mM tempol and different concentrations of superoxide generators. The reactions were performed for 3 h at 30 or 37 °C and then quenched with 100 μl methanol, and the supernatant was analysed using LC–MS.
For in vivo studies, 100 ml of culture was inoculated with a preculture (1 ml) of E. coli BL21(DE3) expressing EasCCf. Protein expression was induced by 5 μM isopropyl β-d-thiogalactopyranoside. The cells were then collected by centrifugation at 5,000 rpm for 10 min and resuspended in a fresh LB medium (5 ml) containing 50 µg ml−1 of kanamycin. Each 50-μl reaction contained 40 μl cell suspension, 0.5 mM substrate, 2 mM NADPH, 240 mM Thiourea and different concentrations of plumbagin and clofazimine. The mixtures were incubated at 30 °C for 12 h and quenched with 100 μl methanol. The resultant supernatant was analysed using HPLC.
ITC tests
MicroCal iTC200 was used for the characterization of the interaction between EasCCf and PCC. EasCCf was diluted in an ITC buffer (150 mM NaCl and 20 mM Tris-HCl (pH 7.5)) to a final concentration of 20 μM. PCC and NADPH were dissolved in the ITC buffer with both in a final concentration of 200 μM. The ITC conditions were as follows: 330 μl of the enzyme was first injected into the sample cell. Titration was then initiated with a first injection of 0.4 μl of the corresponding substrate, followed by 19 injections of 2 μl at 25 °C. Blank tests were carried out using an ITC buffer instead of enzymes in the titration step and were used for baseline correction. Instrument software was used to calculate the normalized heat released from each injection.
Measurement of H2O2 generated in EasC reaction
OxiVision Green hydrogen peroxide sensor (AAT Bioquest) was used to quantitatively determine the H2O2 generated in EasC reaction according to the manufacturer’s instructions. The 50-μl EasCcf reaction solution containing 50 mM Tris-HCl buffer (pH 7.5), 10 μM enzyme, 0.6 mM substrate and 2 mM NADPH was first incubated at 30 °C for 1 h. Then, the preprepared 50-µl sensor solution containing 20 mM HEPES buffer and 10 µM OxiVision Green H2O2 sensor was added into the reaction. Finally, a total of 100 µl mixture was incubated in a 96-well plate at room temperature, and fluorescence intensity was recorded at Ex/Em = 490/525 nm for 15–75 min using an EnSpire 2300 Multimode Reader (PerkinElmer). A 50-μl reaction solution without enzyme was used as the negative control; 1 mM H2O2 was diluted by ratio and used to prepare the standard curves of H2O2 according to the manufacturer’s instructions.
Stopped-flow spectrophotometry
The sequential mixing stopped-flow apparatus (model SX20), equipped with a dedicated computer system and software, was sourced from Applied Photophysics. A monochromator and a diode-array detector (Applied Photophysics PD.1), integrated with the stopped-flow machine, were used to track the progress of all reactions. All measurements were conducted at 25 °C in 50 mM phosphate buffer (pH 7.5). The diode-array detector was specifically used for these measurements, and each sample was subjected to at least three independent determinations.
Under aerobic conditions, an experiment was designed to investigate whether binding of the substrate PCC to EasCCf could facilitate the acquisition of electrons on the haem iron centre and subsequent oxygen binding. One syringe was loaded with 40 µM EasCCf dissolved in 50 mM phosphate-buffered saline (PBS) at pH 7.5, whereas the second syringe contained 0.5 mM PCC.
To gain insights into the intermediate iron–oxygen complex formed during the EasCCf reaction, a stopped-flow apparatus was harnessed to monitor UV–Vis spectral changes occurring in 1 s under aerobic conditions. In this experiment, the first syringe contained 40 µM enzyme in 50 mM PBS at pH 7.5, whereas the second syringe contained a mixed solution comprising 0.5 mM PCC.
EPR measurement
EPR spectra were acquired using a Bruker EMXplus spectrometer equipped with a helium-flow cryostat operating at a temperature of 15 K. The experimental parameters were set as follows: microwave frequency of 9.406 GHz, microwave power output of 20 mW, modulation frequency adjusted to 100 kHz and modulation amplitude of 20 G. Spectral scans were conducted over a broad range, from 5 to 505 mT.
To maintain anaerobic conditions, oxygen was removed from the buffer solutions using nitrogen displacement, and the enzymes and reagents (sodium dithionite, PCC and CC) were treated anaerobically for 3 h in a glove box. Samples were diluted to 200 μl with anaerobic buffer and transferred to EPR tubes for analysis. Four groups of samples contained 492 μM purified EasCCf, 442 μM EasCCf and 10 mM sodium dithionite, 480 μM EasCCf and 5.19 mM PCC, and 480 μM EasCCf and 4.93 mM CC, respectively.
Circular dichroism measurement
Circular dichroism spectra were collected using a circular dichroism spectrometer (Chirascan; Applied Photophysics) to investigate the conformational stability of protein EasCCf. Far-UV spectra (spanning 180–260 nm) were recorded in a 0.1-cm path length quartz cuvette with an internal volume of 0.2 ml. The protein was diluted to a concentration of 0.967 mg ml−1 in 10 mM phosphate buffer at pH 7.0.
To elucidate the role of NADPH in the EasCCf reaction, circular dichroism spectra were analysed for EasCCf that reacted with PCC in the presence and absence of the cofactor NADPH. Specifically, the reaction mixture containing NADPH comprised 35 μM EasCCf, 1 mM PCC and 10 mM NADPH, whereas the mixture lacking NADPH contained only 35 μM EasCCf and 1 mM PCC.
After incubating these reaction systems for 2, 4 and 8 h, the reactions were halted by transferring the systems to ice. By comparing the circular dichroism spectra obtained at these time points and under varying conditions (with or without NADPH), insights into the effect of NADPH on the conformational stability and reaction mechanism of EasCCf with PCC could be gained.
O18-labelled superoxide assay
To produce O18-superoxide used for the EasCCf reaction, 50 mM PBS buffer (pH 7.5), 2 mM NADPH and 8 mM SOTS-1 were added to an 18O2-containing tube in a glove box. After incubation for 10 min at room temperature, 50 μl of the mixture was transferred into a 1.5-ml centrifuge tube in open air and mixed thoroughly using a vortex oscillator to release the residual 18O2. Then, 0.6 mM PCC and 40 μM EasCCf were added to the mixture, and the reaction was further performed at 30 °C for 1 h. The negative control, without SOTS-1, was prepared as described above. The reactions were quenched with 100 μl methanol, and the supernatant was then analysed using HRMS.
Reporting summary
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