Cryo-EM structure of the yeast Saccharomyces cerevisiae SDH provides a template for eco-friendly fungicide discovery

Subject terms: Enzymes, Proteins, Cryoelectron microscopy

Purification and characterization of ScSDH

Native protein complexes isolated under physiologically relevant conditions are regarded as superior experimental specimens for high-resolution structural analysis. SDH is classified as a membrane protein, which presents significant challenges in its extraction and purification. To elucidate the three-dimensional architecture of ScSDH in its functional state, the enzyme was isolated from its native cellular environment through endogenous expression systems according to the method described by Graham²⁴ . The mitochondrial membrane was collected by mechanical homogenization and differential centrifugation, and then further purified by ion exchange chromatography and gel filtration chromatography (Supplementary Fig. 1a). Blue native polyacrylamide gel electrophoresis (BN-PAGE, Supplementary Fig. 1b) showed that a single bond is present. SDS-PAGE (Supplementary Fig. 1c) and Liquid Chromatography Mass Spectrometer (LC-MS, Supplementary Table 1) successfully identified the four subunits of ScSDH, including SDHA, SDHB, SDHC, and SDHD. It confirmed that the purified protein was ScSDH. The activity of the purified ScSDH was further verified by the SDH activity assay method, and its K_m was 195.13 ± 8.09 μM, k_cat was 58.90 ± 0.36 min⁻¹ (Supplementary Fig. 1d, Supplementary Table 2). The above results indicated that the SDH extracted from S. cerevisiae is a functional complex.

The overall architecture of ScSDH

Cryo-EM has been successfully utilized for structural analysis of membrane proteins in the fields of pesticides and pharmaceuticals, contributing to drug target discovery and mechanism research^25–27 . Here, the ScSDH was determined by cryo-EM, with a total resolution of 3.36 Å (Fig. 1, Supplementary Fig. 2, Supplementary Table 3). ScSDH adopts a mushroom-like architecture, comprising a hydrophilic head (SDHA and SDHB subunits) and a hydrophobic membrane anchor (SDHC and SDHD subunits). All subunits assemble into a monomer, similar to porcine SDH⁷ .

The SDHA subunit consists of four domains, flavin adenine dinucleotide (FAD)-binding domain, capping domain, helical domain, and C-terminal domain. The EM- density of FAD could be observed in the SDHA subunit, bound to the FAD-binding domain (Fig. 1b). The capping domain displays poorer density than the overall structure, likely due to its flexible loop-rich composition. Notably, the capping domain of ScSDH exhibits greater amino acid flexibility than other eukaryotic SDHAs, and this flexibility may arise from substrate or ligand binding at the FAD-capping domain interface^28,29 . Adjacent to SDHA lies the butterfly-shaped SDHB subunit, housing three iron-sulfur clusters ([2Fe-2S], [4Fe-4S], [3Fe-4S]) across two domains (Fig. 1c). Like porcine SDHB, the [2Fe-2S] cluster is coordinated by an N-terminal loop and the others reside in the C-terminal domain, bound by conserved cysteines typical of SDHs. Overall, the soluble head (SDHA and SDHB) shares secondary structure features with known bacterial and mammalian SDHs^7,11,30 .

The hydrophobic transmembrane anchor tail is situated opposite the hydrophilic head of SDH and contains two subunits, SDHC and SDHD (Fig. 1d). SDH diversity primarily stems from transmembrane regions, where subunit count (1, 2, 3), prosthetic groups (heme b, Fe-S cluster), and heme numbers vary (0, 1, 2)³¹ . In the realm of eukaryotic SDHs, the structural characteristics of membrane domain are broadly conserved. In ScSDH, both transmembrane subunits traverse the membrane three times. Unlike other eukaryotic SDHs, no clear heme density was observed between transmembrane subunits. This is consistent with the results of heme is nonessential for SDH function^32,33 .

Phosphatidylethanolamine (PE) molecule was identified through density mapping, a finding that was corroborated by subsequent mass spectrometry analysis (Supplementary Fig. 1e). This PE unit was structurally located between the transmembrane helices II and III on the cytoplasmic face, with its tail extending into the membrane. In contrast, cardiolipin (CL), despite being detected by mass spectrometry, was conspicuously devoid in the map density profile. This interesting omission suggested that CL may be involved in highly dynamic structural interactions within protein complexes, thus avoiding traditional density mapping techniques. A parallel phenomenon also occurred in M. smegmatis SDH (PDB ID:7D6X)¹² .

Heme is absent in ScSDH

The SDH superfamily comprises two enzymes—SDH and QFR (short for fumarate reductase)—that catalyze reversible metabolic reactions³⁴ . These enzymes are characterized by their composition of four structurally analogous subunits. The SDH superfamily proteins are classified into six subtypes based on the number of transmembrane subunits and the heme content within the transmembrane domain. Previous work suggested that ScSDH is of type C³⁵ . However, the structure of ScSDH resolved in this study revealed a lack of heme density, indicating that ScSDH was classified as type D, which differed from the SDH of most eukaryotes.

The cryo-EM structure of ScSDH provided a high-resolution density map of the transmembrane region, allowing for clear visualization and comparison of various amino acids and their side chains. A key structural difference between porcine SDH and ScSDH involves residue D108: tyrosine (Tyr_SDHD108) in ScSDH, which replaces histidine (His_SDHD102) in porcine SDH (Fig. 2). This substitution makes the distance between Tyr_SDHD108 and His_SDHC156 less than 4 Å, creating spatial hindrance that precludes heme binding. To verify the lack of heme in ScSDH, we measured the absorption spectra of ScSDH and porcine SDH. As shown in Fig. 3a, the reduced state of porcine SDH exhibits a characteristic absorption peak of reduced heme at 560 nm. No heme absorption peak was observed in either the oxidized or reduced state of ScSDH, but when heme was added, a heme absorption peak could be observed. To investigate the effect of heme deficiency on SDH activity, we detected ScSDH activity in the presence and absence of heme (Supplementary Fig. 1f), confirming that ScSDH maintains normal SDH activity. It is worth noting that in the presence of substrate ubiquinone-1 (UQ1), the k_cat value of ScSDH significantly increased by about 10 times (Supplementary Table 2), while other species only increased by about 2 times, indicating that the ScSDH has excellent quinone mediated electron acceptance ability^11,12 . The structural differences, spectral results, and activity detection collectively confirm that there is no heme in ScSDH and the absence of heme does not affect the activity of SDH enzyme.

The result that ScSDH lacks the heme contradicts long-standing biochemical assumptions^33,36 . Early models were constructed based on sequence homology with heme-containing homologous proteins, assuming that Cys_SDHD109 forms a disulfide bond with porphyrin, while the adjacent Tyr_SDHD108 was modeled as an outward-facing conformation. Cryo-EM structural data revealed shows the opposite: Tyr_SDHD108 adopts an inward conformation, blocking the heme pocket the heme pocket (Fig. 2, Supplementary Fig. 3), which explains why heme is not bound in ScSDH.

Within the vast and varied expanse of the SDH superfamily proteins, the heme content within the transmembrane region has long been considered as a defining characteristic. However, recent findings have revealed that there are some rare exceptions to this rule. Two instances have been documented where the transmembrane segment lacks heme: the E. coli QFR (PDB ID: 1L0V)³⁰ and the T. thermophila SDH (PDB ID: 8B6G)¹⁴ . The distinctiveness of E. coli QFR was marked by the absence of heme in its transmembrane region with an additional electron density, suggesting an alternative molecular occupancy that facilitates electron transfer between the quinone-binding sites Q_p (a proximal quinone binding site) and Q_d (a distal quinone binding site)³⁰ . Moreover, cryo-electron tomography has revealed the structure of SDH in T. thermophila, where it is integrated as a component of a supercomplex¹⁵ . Despite the absence of heme in the transmembrane region, the soluble subunit SDHTT1 on the matrix side contained a bis-histidine C-type heme covalently bound by a single cysteine residue. This finding underscored the intricate relationship between protein structure and heme binding within the SDH superfamily.

Electron transfer pathway in ScSDH

All the prosthetic groups within the structure of ScSDH, including FAD, [2Fe-2S], [4Fe-4S], and [3Fe-4S] clusters, were unambiguously resolved in the cryo-EM density map (Fig. 3b, Supplementary Fig. 4g). The edge-to-edge distances between these redox-active prosthetic group, which were less than 14 Å, fell within a range conducive to efficient electron transfer. FAD was covalently anchored to the SDHA subunit, assuming the pivotal role of the initial electron acceptor within the complex. The catalytic activity of the complex was mediated by FAD, facilitating the oxidation of succinate and the concomitant reduction of fumarate³⁷ . To gain a more intuitive understanding of electron transfer within SDH and to elucidate the reduction process of quinone, we determined a structural analysis of the complex formed by ScSDH and UQ1.

The quinone binding site was constructed by the highly conserved B chain, D chains, and the more variable C chain, exhibiting structural similarity to the Q_p sites of human and porcine (Fig. 3c). The UQ1, the final electronic receiver, through an intricate hydrogen-bonding network, principally involving Trp_SDHB194 and Tyr_SDHD120, thus catalyzing the electron transfer essential for the metabolic vigor of the cell. The short distance (6.6 Å) between UQ1 and the [3Fe-4S] cluster facilitates rapid electron transfer.

In E. coli, porcine and human systems, succinate oxidation liberated electrons that were efficiently channeled towards ubiquinone through a complex redox cascade, comprising FAD, [2Fe-2S], [4Fe-4S] and [3Fe-4S] clusters. The final step in this electron transfer cascade was the delivery to ubiquinone, a process accelerated by heme b, which acted as a central electron pool^38,39 . The strategic formation of semi-quinone at this stage was a key enhancement, enhancing both the rate and efficiency of electron transfer, thereby underscoring the intricate orchestration of biological energy conversion⁴⁰ . In the SDH structure of M. smegmatis, Rieske type [2Fe-2S] replaced heme b as the electron pool, rather than completely lacking this type of cofactor¹² . The structural conservation and redox center alignment between ScSDH and SDHs from E. coli, M. smegmatis, porcine and human sources suggested a commonality in their electron transfer mechanisms. Yet, the absence of heme b in ScSDH, as indicated by structural features and spectral experimental data, pointed to a more direct electron transfer pathway. Structural analyses further reveal that the electron transfer distance from [3Fe-4S] cluster to UQ1 in ScSDH (6.6 Å) is shorter than in porcine, M. smegmatis SDH1, and E. coli systems, promoting enhanced efficiency. While identical to human SDH in distance, the UQ1 of ScSDH adopts a distinct conformation stabilized by hydrogen bonding with Trp_SDHB194, diverging from human UQ1 binding. This finding challenges the earlier notion that heme b is a ubiquitous component of SDHs, reinforcing the idea that it is not an essential structural element for quinone reduction—a conclusion further supported by the ScSDH structure.

We propose heme absence in ScSDH reflects adaptation to its fermentative lifestyle, which reduces oxygen demand compared to other yeasts. Consequently, ScSDH evolved enhanced hypoxia tolerance, eliminating the need for the antioxidant role of heme.

Structural insights into the Q-binding site for fungicide discovery

SDH, a ubiquitous enzyme in mammals, bacteria, plants, and fungi, serves as a critical target in fungicide development. The quinone-binding sites of SDH exhibited species-specific structural diversity due to the non-conservatism of SDHC and SDHD. In porcine SDH and E. coli QFR, two distinct quinone-binding sites, Q_p and Q_d, have been identified, located on opposing sides of the membrane-spanning region^7,30 . In M. smegmatis SDH2 trimer, the classical Q_p site was blocked due to the presence of SDHF, forming the specialized Q_D1 and Q_D2 sites¹³ . However, the Q_p site is still recognized as the classical inhibitor binding site.

PYD, a pyrazole amide fungicide developed by Syngenta, gained global agricultural acceptance since 2017. Its structure, featuring a difluoromethyl group at the pyrazole 3-position and an N-methoxy group, enhanced its control spectrum and potency^41,42 . To elucidate the mechanism of action of PYD, we analyzed the cryo-EM structure of the PYD-ScSDH complex at 3.23 Å resolution (Fig. 4, Supplementary Fig. 5). Our analysis revealed that PYD bound via hydrogen bonds with Trp_194 in SDHB and Tyr_120 in SDHD, and its N-methoxy group formed additional hydrogen bond with Ser_94 in the C chain, enabling it to displace UQ1. Notably, the pyrazole ring of PYD formed a cation-π interaction with Arg_97 in SDHC (Fig. 4a). The bridging moieties of PYD formed an angle about 90°, clearly mapped in the cryo-EM density (Supplementary Fig. 5g).

Further analysis of amino acids within 5 Å of PYD showed that the inhibitor binding pocket is primarily surrounded by conserved B chain residues (Pro_190, Ser_191, Trp_193, Trp_194, His_237, Ile_239) and D chain residues (Asp_119, Tyr_120), along with the more variable C chain (Supplementary Figs. 6, 7). The C chain, a key determinant of structural diversity, includes residues Leu_81, Trp_90, Ser_93, Ser_94, Arg_97, and Ile_98, which are critical for inhibitor binding. Sequence alignment revealed that the inhibitor binding site on the ScSDHC subunit is relatively conserved in pathogenic fungi (Supplementary Figs. 6, 7). In addition, structure superposition also revealed structure similarities of Q_p pocket between ScSDH and the predicted fungal SDHs (Supplementary Fig. 8)⁴³ . This suggests that the ScSDH would be a promising model for guiding fungicides within a certain range.

Previously, the SDH complex from porcine was selected as a primary structural template for SDHI development, with its inhibitor bound complexes being extensively characterized through crystallographic and computational analyses^44–46 . Comparative analysis of inhibitor binding models in porcine SDH revealed the openings of active cavity caused by variations of inhibitor. In porcine SDH, the flexibility of Trp_61 in the C chain emerges as a key factor determining the dimensions of active cavity. Variations in inhibitor size induce distinct angular shifts of the Trp_61 indole ring, enabling cavity size adaptation through dynamic structural rearrangements (Fig. 4b). In contrast, the ScSDH employs Trp_90 for similar functional adjustments. Structural superposition analysis (Fig. 4c) reveals that the ScSDH Trp_90 is spatially aligned with porcine SDH Met_65, which is located inside the cavity. Therefore, the ScSDH Trp_90 is a residue restricted by the proximal amino acid and has limited rotational freedom. This steric restriction results in a constitutively smaller Q_p cavity in ScSDH compared to porcine SDH, highlighting the importance of considering species-specific amino acid compositions and spatial constraints in inhibitor design. The elucidation of the fungal ScSDH structure has established a structural framework for designing antifungal SDHIs with enhanced target specificity and reduced off-target effects in eukaryotic organisms.

Molecular design of SDH fungicide

The PYD was selected as the starting point to design SDH inhibitor. The complex structure of PYD-ScSDH was subjected to the CORE_GEN mode in ACFIS 2.0 tool to obtain a list of core fragments of ligand (Supplementary Fig. 9)⁴⁷ . PYD was spliced into nine fragments and the binding free energy (ΔG) of them with SDH, as well as ligand efficiency (LE), were calculated. LE was a valuable method to measuring the fragment contribution for ΔG (Supplementary Fig. 9). The LE of fragment 1 was measured at 0.42, which rose to 0.53 in fragment 2 following the addition of one methoxy-amide bond. This result indicated that the amide bond in PYD was very important to maintain the activity. Among fragment 1 to 9, the fragment 3 and 4 gave out the higher LE than others fragments. Based on our previous study, the cyclopropyl attached to nitrogen atom would be favorable for increasing the compound activity against porcine SDH⁴⁸ . Here, the fragment 10 and 11 were built based on fragment 3 and 4, respectively. It should be noted that the LE in fragment 10 was the highest with 1.20. Consequently, fragment 10 was selected as the core fragment.

Then, the 3D structure of fragment 10-bound ScSDH was uploaded to CAND_GEN module for fragment growing. At last, ACFIS 2.0 provided a series of fragment 10-derived compounds as potential SDH ligands (Supplementary Fig. 10). Among them, ligand 3 exhibited a lower calculated ΔG than PYD which was inferred to be bioactive toward SDH (Supplementary Table 4). Our previous studies had pointed out that the pyrazole ring attached to fluorine atom in the scaffold of SDH inhibitor was benefit for activity against SDH^48–50 . Consequently, a series of pyrazole carboxamide with N-cyclopropyl compounds were synthesized (Fig. 5a).

All target compounds were evaluated against ScSDH. As shown in Table 1, most compounds exhibited good activity against ScSDH at 1 μM concentration. Notably, compound E8 demonstrated markedly superior potency (IC₅₀ = 0.030 μM), surpassing the reference compound PYD (IC₅₀ = 0.040 μM) by nearly 1.3-fold. These promising results warrant further in-depth biological evaluation of E8.

The inhibition constant (K_i) represents the concentration of free inhibitor at which 50% of the enzyme is bound, and its value is independent of both enzyme and substrate concentrations used in the assay. Consequently, we define the inter-species selectivity of a compound as the ratio of its K_i for porcine SDH and ScSDH. As depicted in Table 2, the K_i of E8 against ScSDH, as well as porcine SDH, was performed. The results exhibited that E8 displayed 0.019 μM and 0.281 μM against ScSDH and porcine SDH, respectively, which were better than PYD. Among ScSDH and porcine SDH, the species selectivity for E8 was 14.78, which decreased to 8.28 for PYD. All these results indicated that E8 showed higher species selectivity than PYD. To investigate the mechanism of action of E8, the cryo-EM structure of E8-bound ScSDH (3.06 Å) was determined (Supplementary Fig. 11). The overall binding mode E8 was similar to that of PYD, forming hydrogen bond with Tyr_SDHD120 and Trp_SDHB194, cation-π interaction with Arg_SDHC97 (Fig. 5b, c). As shown Table 3, the double bond in E8 made the electronic energy (ΔE_ele) of it with ScSDH was more favorable than PYD, resulting in the ΔG_cal of E8 lower than PYD. All these results were consistent with the experiment results.

Fungicidal activity of E8

Compound E8, which demonstrated excellent enzyme inhibitory activity, was systematically tested for its fungicidal activity. In vitro tests against representative plant-pathogenic fungi revealed the broad-spectrum efficacy of E8, with inhibition rates exceeding 70% at a low dosage of 1.56 mg/L, matching or surpassing PYD (Table 4). Spore germination assays demonstrate that E8 significantly inhibits the germination of 4 representative pathogenic species from different subphyla, confirming that this inhibitor impairs pathogenicity by blocking conidial germination (Supplementary Table 5). In vivo greenhouse experiments further demonstrated the significant activity of E8 against wheat powdery mildew (WPM) and cucumber powdery mildew (CPM) with EC₅₀ values for E8 at 0.0462 mg/L for WPM and 0.5431 mg/L for CPM (Supplementary Table 6). Notably, E8 exhibited exceptional efficacy against WPM, showing over 20-fold greater activity than PYD. At low dose (0.097 mg/L), PYD-treated leaves exhibited numerous symptoms, whereas E8-treated showed significantly fewer symptoms, highlighting the superior efficacy of E8 even at lower application rates (Supplementary Fig. 12). These 12 plant-pathogenic fungi species span 3 subphyla, which suggests that diverse fungi species are susceptible to the E8.

Field trials served as an effective approach to evaluate the control efficacy of compound under conditions approximating actual agricultural production. These trials assessed E8 against WPM, and fusarium head blight (FHB), a significant soil-borne disease that has adversely affected wheat yields and quality in recent decades⁵¹ . For WPM, the control effects were 92.38% for E8 with 187.5 g a.i./ha dosage, which was similar to PYD (93.35% control effects) (Supplementary Table 7). For FHB, E8 showed higher efficacy (88.81% control effect) at 150 g a.i./ha than that of validamycin (48.42% control effect), compared to tebuconazole (84.45% control effect) and PYD (89.08% control effect) (Fig. 6a, Supplementary Table 8).

The management of FHB present a dual challenge: controlling the disease itself and reducing the levels of deoxynivalenol (DON) produced by Fusarium species⁵² . As a result, lowering DON levels had become a critical standard for assessing the success of FHB management strategies. Significantly, E8 reduced DON levels and enhanced wheat yield (Supplementary Table 9). In the absence of fungicides, FHB can produce DON levels up to 1559.5 μg/kg. After applied E8, it reduced DON to 165.3 μg/kg at 180 g a.i./ha dosage (89.40% DON inhibition) and to 217.5 μg/kg at 150 g a.i./ha (86.05% DON inhibition), comparable to PYD (Fig. 6b). Additionally, E8 increased wheat yield by 10.18%, surpassing PYD (9.49%). These results highlight the potential of E8 as an SDH inhibitor for further development, effectively controlling FHB, reducing DON content, and enhancing yield (Fig. 6c, d).

The aquatic toxicity of a compound critically determines its developmental potential. Regulatory frameworks classify compounds with a LC₅₀ (96 h) ≤ 1.0 mg a.i./L as highly toxic to aquatic organisms, imposing stringent restrictions on their use⁵³ . While the reference compound PYD exhibits high toxicity (LC₅₀ (96 h) = 0.729 mg a.i./L), precluding its application in rice fields, compound E8 demonstrates significantly reduced toxicity. Comprehensive evaluation classifies E8 as moderately toxic to zebrafish (LC₅₀ (96 h) = 1.01 mg a.i./L) (Supplementary Table 10), indicating enhanced environmental compatibility. The structural comparison between PYD and E8 revealed a carbon-carbon double bond in E8. Previous analysis showed that similar carbon-carbon double bonds also exist in other agrochemicals, such as silthiofam and imazalil, and their carbon-carbon double bonds tend to be converted into dihydroxy structures during metabolism^54,55 . Therefore, we postulated that the double bond of E8 may generate dihydroxy-containing metabolites in vivo or in environmental systems. The ecological structure-activity relationships model (ECOSAR) predicted that the proposed dihydroxy metabolite would exhibit low acute toxicity to fish (LC₅₀ (96 h) = 344 mg/L) (Supplementary Fig. 13). This result is consistent with the reduced aquatic toxicity profile of E8, indicating that the double bond serves as a key structural determinant for detoxification in aquatic organisms.

In summary, the compound E8 has shown remarkable efficacy in fungicidal activity, as evidenced by greenhouse pot experiments and field trials, while also markedly decreasing toxicity to aquatic organisms. This makes it a highly promising candidate for a SDH inhibitor. Its strong inhibitory effects on various plant-pathogenic fungi, broad-spectrum fungicidal activity, and impressive results in field trials, along with its minimal toxicity to aquatic life, indicated that E8 has the potential to be a significant asset in future crop disease management.

In this work, a comprehensive structural analysis of ScSDH is present. The absence of heme in ScSDH and the identification of its electron transfer pathway underscore the diversity within the SDH superfamily and emphasize the importance of species-specific structural variations in inhibitor design. The structural insights into the quinone-binding site of ScSDH, provided a blueprint for developing SDHIs. The identification of E8, demonstrated the utility of ScSDH as a template for rational fungicide design, and offered practical solutions to address fungal resistance and environmental toxicity in modern agriculture. By providing a detailed structural framework for the design of more effective and eco-friendly fungicides, this study holds significant promise for enhancing crop protection while minimizing environmental impact, thereby playing a crucial role in promoting sustainable agricultural development. Future research should focus on exploring the structural diversity of SDH in other fungal species and optimizing inhibitors for practical agricultural applications.

Yeast strain and culture

Saccharomyces cerevisiae strain, red star, cultured in YPG media (1% yeast extract, 2% tryptone and 3% glycerol) at 30 °C to an OD₆₀₀ of 1.5 and then harvested by centrifugation at 3000 ×ばつ g for 5 min. Cell pellets were frozen at −80 °C until use.

Isolation of mitochondria

The cell pellets were thawed and suspended in buffer A (50 mM Potassium phosphate, pH 7.4, 1 mM EDTA, 0.9% KCl and 0.5 mM PMSF). Cells were lysed by high-pressure crusher at 1350 bar and then centrifuged at 3000 ×ばつ g for 10 min to remove cell debris. The pH of supernatant was adjusted to 5.4 with 1 M glycine-HCl, pH 2.35 and then centrifuged at 12,000 ×ばつ g for 45 min to collect crude mitochondrial pellets. And then pellets were resuspended with buffer B (100 mM Potassium phosphate, pH 7.4, 1 mM EDTA, 0.5 mM PMSF and 0.25 M sucrose) and centrifuged at 12,000 ×ばつ g. The pellets were identified as mitochondria and stored in a −80 °C.

Protein purification and characterization

The purification of ScSDH was carried out according to the method proposed by Graham²⁴ . The isolated mitochondrial pellets were thawed and suspended in buffer B with homogenized. Mitochondrial membrane proteins were extracted with 1.6% (w/v) of the detergent sodium cholate for 3 h at 4 °C. Ammonium sulfate gradient salting out was used to purify ScSDH. ScSDH was precipitated at 45% to 60% ammonium sulfate saturation. The precipitate was resolubilized in buffer C (100 mM Potassium phosphate, pH 7.4, 1 mM EDTA and 0.5 mM PMSF) and diluted with buffer D (20 mM sodium phosphate pH 7.4, 0.1% DDM). The sample were loaded onto a Source 15Q column (GE Healthcare). The ScSDH was eluted from Source 15Q in buffer D with 200 mM KCl. The fractions contain ScSDH were incubated with inhibitor and then loaded onto a Superdex 200 increase column (GE Healthcare), which had been equilibrated in ×ばつPBS (Gibco) with 0.05% DDM. Elution was performed at a flow rate of 0.5 mL min⁻¹. The peak fraction was collected and concentrated to 6 mg/mL.

The purified ScSDH were characterized with 4–16% BN-PAGE gel (Invitrogen), 15% SDS-PAGE and LC-MS^56–58 .

Characterization of enzyme activity

The activity of SDH was characterized using the 2,6-dichlorphenolindophenol (DCIP, Sigma) method^11,48 . Ubiquinone-1 (UQ1, MCE) was selected as the final electron acceptor to assess succinate dehydrogenase activity. The reaction system consisted of 20 mM phosphate buffer, 15 μM DCIP, 200 μM UQ1 and 0.01 to 1.5 mM succinate. The enzyme activity assay was performed at 30 °C by using the Cytation 5 (Biotek).

For the enzyme activity comparison of ScSDH with or without heme. The reaction system consisted of 20 mM phosphate buffer, 30 μM DCIP, 20 mM succinate, 0.394 μM heme (Sigma) and 0.05 to 1.0 mM UQ1.

Mass spectrometry

To identified the four subunits of ScSDH, following SDS-PAGE separation, 5-mm wide strip gel lanes were excised. The gel strips were washed in ddH₂O and discolored using 50% acetonitrile (Sigma). Then, disulfide bonds were reduced using 20 mM dithiothreitol (Sigma) for 1 h at 55 °C, and subsequently free sulfhydryl groups were carbamidomethylated using 20 mM iodoacetamide (Sigma) for 30 min at room temperature in the dark. Trypsin (Promega) was added to digest samples at 37 °C for 14 h. The peptide samples were extracted using 65% acetonitrile and 5% formic acid in ddH₂O and then dried under vacuum. Finally, Samples cleaned up using Oasis HLB cartridges (Waters) and extracted with 80% acetonitrile and dried. For LC-MS/MS Analysis, the dried peptide samples were redissolved in 30 μL of 0.1% formic acid in ddH₂O (Thermo Scientific) and transferred into injection vials. Using an Easy nLC 1200 Rapid Separation Liquid Chromatography system (Dionex/Thermo Scientific, Germany), 3 μL of each sample were trapped on a C18 PepMap100 precolumn (particle size 3 μm; Dionex/Thermo Scientific) and analytical column (C18; particle size 2 μm; Thermo Scientific, Germany) with an aqueous-organic gradient (eluent A: 0.1% (v/v) formic acid in ddH₂O; eluent B: 0.1% (v/v) formic acid/80% (v/v) acetonitrile/19.9%(v/v) ddH₂O; The gradient was set as follows: 0–3 min, 8–10% B; 3–45 min, 10–25% B; 45–54 min, 25–45% B; 54–56 min, 45–99% B; 56–60 min, 99%B. Eluting peptides were electrosprayed (2.1 kV; transfer capillary temperature 275 °C) in positive ion mode into an Orbitrap Exploris480 tandem mass spectrometer equipped with a Nanospray. Total acquisition time 60 min according to the gradient. FT full MS (m/z 350 to 1200; resolution 60000 FWHM) and ACG Target 300, RF Lens 50%, Mass width ± 10 ppm, Isolation Window 1.6 m/z, HCD Collision Energies 30, Exclusion duration 45 s. For protein identification, Peak lists were extracted from fragment ion spectra using the Proteome Discoverer (PD) 3.0 (Thermo Scientific). Data were searched with Sequest HT against the red star protein database, including the sequence of SDH subunits. For each dataset, Acetyl (protein N terminus) and oxidation (Met) were chosen as variable modifications, Carbamidomethylation on cysteine (+ 57.021 Da) was specified as static modification. fragment mass tolerance was set to ±10 ppm, and two missed cleavage was allowed. high confidence corresponding to a false discovery rate (FDR) ± 1%.

Co-purified lipids from ScSDH were extracted by chloroform/methanol (2:1, v/v) and dried. Then the sample was re-dissolved in 60% acetonitrile. Analysis of samples was conducted on a SCIEX ExionLC system coupled with a SCIEX ZenoTOF 7600 mass spectrometer. Lipid analysis was carried out using reverse-phase (RP)-LC–MS in both positive and negative ESI modes. The separation was performed on a Kinetex C18 column (2.6 μm particle size) maintained at 45 °C with a flow rate of 0.30 mL/min. HPLC buffer A consisted of acetonitrile, methanol, and water (1:1:1, v/v/v) containing 5 mM ammonium acetate, while buffer B consisted of 100% isopropanol with 5 mM ammonium acetate. The gradient was set as follows: 0–0.5 min, 20% B; 0.5–1.5 min, 20–40% B; 1.5–3 min, 40–60% B; 3–13 min, 60–98% B; 13–14.5 min, 98% B; 14.6–17 min, 20%B; 1.5–3 min, 40–60% B. A 2-μL resultant solution was injected for LC–MS/MS analysis. MS/MS analysis was performed using the following parameters: ion spray voltage, 5.5 kV (+) and 4.5 kV (−); curtain gas, 35 psi; gas 1 and gas 2, 55 psi; declustering potential, 80 V (+/−); and interface heater temperature, 550 °C. Collision energy was set to 45 ± 20 V in negative modes, with dynamic background subtraction applied. The experiments were conducted with a 150 ms accumulation time for TOF MS (m/z 200–1200) and a 25 ms accumulation time for TOF MS/MS (m/z 70–1200) using information-dependent acquisition mode. MS-DIAL (Version 5.1) was employed to filter and identify candidate lipids using exact mass, retention time, and MS/MS fragmentation patterns. The SCIEX OS workstation (Version 3.3) was applied to verify MS/MS information and quantify lipid peak areas.

Spectroscopy

The buffer solution consisted of 20 mM PBS (pH 7.4), while the enzyme (ScSDH and porcine SDH) was utilized at a concentration of 8 μM. To initiate the experiment, the buffer and enzyme were thoroughly mixed to ensure homogeneity, following the light absorption in the range of 400 nm to 600 nm was monitored, which the air-oxidized signal was obtain. Subsequently, the reductant dithionite was introduced, which the dithionite-reduced signal was obtain.

Electron microscopy sample preparation and imaging

4 μL of 6.0 mg/mL ScSDH were applied to glow-discharged 300 mesh ANTcryoTM R1.2/1.3 grids. The grids were blotted 5 s with force 3 at 8 °C and 100% humidity. The images were collected on a Titan Krios G4 microscope operated at a voltage of 300 kV with a Falcon4 direct electron detector. Single-particle data acquisition was conducted with EPU package, with a calibrated magnification of ×ばつ165,000 which yields a final pixel size of 0.74 or 0.75 Å. A total dose on the detector was approximately 48.94, 49.87, 50.11, and 40.48 electrons per Å2 for ScSDH, ScSDH-UQ1 bound, ScSDH-PYD bound, and ScSDH-E8 bound. Each micrograph stack contains 32 frames.

Imaging processing

The images were processed using software CryoSPARC⁵⁹ . The patch-based motion correction and CTF estimation were conducted using the Patch CTF algorithm. The particles were automatically picked and extracted with a box size of 384 pixels in CryoSPARC. The extracted particles were classified and screened with several 2D classification. Then Ab-initio reconstruction and heterogeneous refinement were performed with all selected particles. Well-sorted particles were finally subjected to non-uniform refinements, resulting in the generation of final maps. The resolution was estimated using the Fourier shell correlation (FSC) 0.143 criterion. All dataset processing can be found in Supplementary Figs. 2, 4, 5, 11 and Supplementary Table 3.

Model building and refinement

The ScSDH atomic model was constructed manually built in Coot⁶⁰ using the predicted structures with AlphaFold. The real-space refinement of the model was carried out using Phenix with Ramachandran restraint⁶¹ and further manual adjustment was performed in Coot. The stereochemistry and geometry of the structure were evaluated using Molprobity⁶² . All reported resolution was estimated using the Fourier shell correlation (FSC) 0.143 criterion.

All figures were generated using the program UCSF Chimera⁶³ , ChimeraX⁶⁴ , and PyMOL (http://pymol.org).

Molecular design of PYD analogs

The PYD-bound ScSDH was set as the starting point to design SDH inhibitor through pharmacophore-linked fragment virtual screening (PFVS) strategy⁶⁵ . The structure-based optimization design of SDH inhibitor was performed with ACFIS 2.0 tool⁴⁷ . The CORE_GEN mode was used to obtain a list of computational core fragment in PYD based on the structure of PYD-bound ScSDH. Fragment 10, which had the largest increment in LE (ligand efficiency), was selected as the core fragment. Then, the three-dimensional structure of fragment 10-bound ScSDH was upload, followed by the CAND-GEN mode to grow ligand. At last, the ACFIS 2.0 provided a series pyrazole carboxamide with N-cyclopropyl compounds as potential SDH inhibitor. Among them, compound 3 exhibited a lower calculated binding free energy than PYD, indicating high potency against SDH. Based on our previous study, a series modified compound 3 were synthesized and preformed hit-to-lead optimization and eventually obtain compound E8.

Chemicals and instruments

All reagents and solvents were purchased from commercial suppliers and used without further purification. The reactions were performed under atmosphere. Reaction progress was generally monitored by thin-layer chromatography (TLC). Intermediates and target compounds were purified by silica gel column chromatography. ¹H NMR, ¹³C NMR, and ¹⁹F NMR were obtained on a Varian Mercury-Plus 600 or 400 spectrometer (Varian Inc., Palo Alto, CA, USA), using chloroform (CDCl₃), or dimethyl sulfoxide (DMSO) - d₆ as deuterated solvents. The NMR data were analyzed using MestReNova 14.0.0 software. Melting points were measured on a BüCHI B-545 melting point apparatus without correction. High-resolution mass spectra (HRMS) was performed with an Agilent 6224 TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA). The detailed synthetic method and characterization data of compounds E1-E10 (¹H NMR, ¹³C NMR, ¹⁹F NMR, and HRMS) can be found in the Supplementary Figs. 14–53 and Supplementary Note 1.

Fungicidal activity in vitro

The in vitro fungicidal activity of compound E8 was evaluated in mycelia growth inhibition method. Sclerotinia sclerotiorum, Fusarium graminearum, Fusarium pseudograminearum, Fusarium oxysporum, Ustilaginoidea virens, Alternaria alternate, Alternaria solani, Phytophthora infestans, Phytophthora capsica, and Exserohilum turcicum were taken as the test objects. The commercial fungicides PYD was chosen as positive control. Fungal discs (5 mm diameter) were punched from the margins of colonies grown for 4 consecutive days. These discs were inoculated onto PDA plates containing a fungicide concentration of 1.56 mg/L, with three replicates per treatment. The plates were then incubated at 25 °C in darkness. The diameters of both the control and treated colonies were measured using the cross method, and the inhibition rate was calculated as Eq. 1:

Spore germination

To evaluate spore germination, fresh spore suspensions were evenly spread onto water-agar medium plates containing a series of gradient concentrations of E8 and PYD, with three replicates per concentration. After incubation at 28 °C for 12 h, spore germination rates at different concentrations were quantified. Germination was defined as the emergence of a germ tube reaching half the length of the minor of spore. The inhibition rate of spore germination was calculated using the Eq. 2:

The data were analyzed with DPS (Date Processing System, 9.5) and the EC₅₀ values of the tested compounds were calculated.

Fungicidal activity in greenhouse

The fungicidal activities of E8 against wheat powdery mildew (WPM, Erysiphe graminis) and cucumber powdery mildew (CPM, Sphaerotheca fuliginea) were evaluated in greenhouse at 100 mg/L, 25 mg/L, 6.25 mg/L, 1.56 mg/L, 0.39 mg/L, and 0.097 mg/L. The commercial fungicides PYD was chosen as positive control. E8 and PYD were dissolved in DMF and diluted with distilled water containing 0.1% Tween 80 solution. Then the diluted solutions were sequentially gradient diluted to the test concentration. For each treatment, three potted wheat and cucumber plants of similar growth stages were selected. The solutions were uniformly sprayed on plant leaves. Following a 24-h drying period, S. fuliginea was inoculated onto cucumber leaves by the spore spray method, while E. graminis was inoculated onto wheat leaves by the spore shaking method. The plants were then placed in a suitable environment. The data were analyzed with Statistical Product and Service Solutions (SPSS) and the EC₅₀ values of the tested compounds were calculated. The pot experiment was entrusted to Zhejiang A&F University for completion.

Fungicidal activity in field

The wheat field experiment against WPM (Erysiphe graminis, wheat variety: huaimai 33) and FHB (Fusarium graminearum, wheat variety: yannong 1212) was carried out in Huai’an (Jiangsu Province, China) in a plot with an area of 20 m². The potential control efficacy was determined via the standard method. Compound E8 was prepared as 10% emulsifiable concentrate (EC) and diluted to 150, 187.5, and 225 g a.i./ha for WPM application, while it was diluted to 112.5, 150, and 180 g a.i./ha for FHB application. In the early onset of E. graminis, the wheat was uniformly sprayed with compound E8. For WPM control, PYD (20%, suspension concentrate, SC) and prothioconazole (30%, oil dispersion, OD) were used as a positive control, and water was used as a negative control. For FHB control, PYD (20%, SC), tebuconazole (430 g/L, SC), and validamycin (24%, aqueous solutions, AS) were used as a positive control, while water used as a negative control. Sprays were applied during early and peak anthesis of wheat, with efficacy against WPM and FHB assessed 14 and 17 days after the second spray, respectively. The disease index of WPM and FHB was determined by Eq. 3:

N = 9 when test WPM, N = 7 when test FHB.

The control efficacy was evaluated by Eq. 4:

The field trial was commissioned by the Plant Protection College of Nanjing Agricultural University for organization and implementation.

DON toxin determination methods: In each plot, samples were collected using the five-point sampling method. At each sampling point, 100 mature wheat spikes were harvested. The collected samples were mixed, rapidly air-dried, and threshed, then processed into flour. The deoxynivalenol (DON) content in the flour was quantitatively analyzed through liquid chromatography, with three replicates set for each plot. The inhibition rates were calculated as Eq. 5:

Average thousand grain weight determination methods: Samples were collected using the five-point sampling method in each plot. At each sampling point, 100 mature wheat spikes were collected. The collected wheat grains were mixed, rapidly air-dried, and threshed. Then 1000 wheat grains were randomly selected. 1000 wheat grains were weighed and each treatment was replicated three times. The yield increase rate was calculated as Eq. 6:

Acute toxicity zebrafish

The trial was conducted by Jiangsu Academy of Agricultural Sciences. To ensure animal welfare, all the animal maintenance and procedures were in accordance with recommendations established by the Animal Ethics Committee of Jiangsu Academy of Agricultural Sciences, as well as the guidance document of Code for Pesticide Registration Testing Quality Management and the Test Guidelines on Environmental Safety Assessment for Chemical Pesticides-Part 12: Fish Acute Toxicity Test (GB/T 31270.12-2014). The zebrafish (Brachydanio rerio H-B.) was juvenile (before reaching sexual maturity) and maintained at 22–23 °C on a 16–8 h light-dark cycle. Acute toxicity assessments of compound E8 on zebrafish were determined with a static test. For the definitive test, serial concentrations of 1.20, 1.00, 0.833, 0.694, 0.578, 0.482 mg a.i./L were set. Dechlorinated tap water (DTW) served as a blank control. Both experimental and control groups were set up with one replicate each, with each replicate consisting of 10 zebrafish. When an experiment resulted in varying concentrations with mortalities, the LC₅₀ and the confidence limits (95%) were estimated using DPS.

Predicted toxicity of metabolite

The Ecological Structure Activity Relationships (ECOSAR) program package (Version 2.2) was used to predict the aquatic toxicity of E8 and its metabolite. The SMILES structure was imported into the program to estimate the acute toxicity to fish for chemicals of interest.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Peer review information

Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

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Supplementary Materials

Data Availability Statement

All the EM density map and structure reported in this study have been deposited in Protein Data Bank (PDB; http://www.rcsb.org) and the Electron Microscopy Data Bank (EMDB; https://www.ebi.ac.uk/pdbe/emdb), following the lists below: EMD-62490 and PDB 9KPS for the apo-ScSDH, EMD-62491 and PDB 9KPT for the ScSDH-UQ1, EMD-62495 and PDB 9KQ3 for the ScSDH-PYD, and EMD-63115 and PDB 9LIG for the ScSDH-E8. PDB codes of previously published structures used in this study are 1ZOY, 3AEE, 3AEB and 3AE9 of porcine; 7D6X of M. smegmatis Sdh1, 1L0V of E. coli, and 8B6G of T. thermophila. Source data are provided as a Source data file. Source data are provided with this paper.