Specialized aldo-keto reductases trigger complete degradation of mycotoxin deoxynivalenol

Domain: chemistry
Published: 2026-02-26
Editorial outcome: major_revision

Abstract
The mycotoxin deoxynivalenol (DON) poses severe threats to human and animal well-being globally. Enzymatic degradation is the most effective way to eliminate DON toxicity, yet no catalytic process for complete degradation of DON has been uncovered. Here, we show that a metabolic pathway initiated by C3-epimerization and C8-reduction is responsible for complete degradation of DON in the DON-metabolizing bacterium Nocardioides sp. S5-5. Two horizontally transferred aldo-keto reductase genes, DONepi and DONrd, have evolved to orchestrate C3-epimerization and C8-reduction respectively. Notably, the octameric-structured DONepi alone catalyzes C3-epimerization of DON by steering the rigid-body rotation of the transient 3-keto intermediate for stereoinverting reduction. Moreover, DONrd can catalyze the C8-reduction of DON and its C3-epimerized product 3-epi-DON simultaneously to form C8-hydroxyl products, which facilitates the further degradation by a potential oxidase and other putative enzymes. DONepi expression in transgenic plants confers resistance to DON, representing potential for controlling mycotoxin contamination pre- and postharvest.

Introduction
Mycotoxins, which are fungal secondary metabolites, contaminate 60-80% of crops globally, posing severe threats to both humans and livestock1. Deoxynivalenol (DON) is the most common mycotoxin worldwide, occurring at higher levels in Europe and Asia2,3. Recently, due to climate change and crop residue incorporation, the increasing frequency of Fusarium head blight epidemics, which is caused by DON-producing pathogenic Fusarium, has led to more serious and recurrent risks of DON contamination in cereal raw materials and finished food products4-6. With stable physicochemical properties, DON cannot be efficiently removed during food and feed processing and can, therefore, enter human and animal food chains7. DON can interfere with the protein translation through binding to the A-site of the peptidyl transferase center on the large subunit (60S) of the eukaryotic ribosome, leading to acute and chronic mycotoxicosis in humans and animals8,9. Microbial and enzymatic degradation represent promising approaches for managing DON contamination due to their high specificity, cost-efficiency, and environmental friendliness10. In the past few decades, several patterns for transforming DON and its structural analogs by diverse microbes and enzymes have been investigated, including: (1) reductive de-epoxidation and glutathione conjugation targeting the C12, 13 epoxide group6,11-14; (2) oxidation, epimerization, and glycosylation targeting the C3 hydroxyl15-17; (3) hydroxylation targeting the C1618; and (4) isomerization targeting the C9-C10 double bond19-21. However, the products resulting from DON transformation via these pathways exhibited varying degrees of retained toxicity22-24.

The final product of DON C3-epimerization, 3-epi-DON, is one of the lowest-toxicity metabolites among the currently known microbial metabolites of DON 12, 23. Enzymes involved in this process have been identified in several bacterial strains, which convert DON to 3-epi-DON through two independent reactions catalyzed by distinct classes of enzymes. The initial step is the oxidation of DON to form the intermediate 3-keto-DON, carried out by pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenases (PQQ-ADHs). Subsequently, the second step entails the reduction of 3-keto-DON to produce the final product 3-epi-DON, which is accomplished by aldo-keto reductases (AKRs) 25,26. Nevertheless, PQQ-ADHs require the particular coenzyme PQQ, which cannot be produced naturally in typical expression systems employed in industrial biotechnology, including E. coli and yeast26. This incapability severely restricts the application of PQQ-ADHs within the realms of food and feed safety, since the need for exogenous addition of the coenzyme PQQ leads to prohibitively high costs. In contrast to the formation of diverse products with differential levels of retained toxicity via the well-known pathway associated with DON transformation, certain microbes have the capacity to degrade DON completely. However, to date, the isolation of strains endowed with such degradation ability has remained scarce. Moreover, the specific enzymes involved in and the precise catalytic mechanisms governing the complete degradation of DON remain unknown.

Here, we identify the enzymes and catalytic process underlying complete degradation of DON in Nocardioides sp. S5-5, a bacterium with the capacity to degrade and assimilate DON. The process is initiated by two distinct pathways: C3-epimerization and C8-reduction. Specifically, two AKR genes, DONepi and DONrd, acquired via horizontal gene transfer mediated by genomic islands, govern the two pathways respectively. To reveal their catalytic mechanisms, we employ Cryo-EM structural analysis and QM/MM metadynamics simulations. Furthermore, in plants, DONepi exhibit substantial activity in increasing the resistance to DON, suggesting the broad application potential in mycotoxin degradation and crop resistance improvement.

Results
To isolate the strain capable of completely neutralizing the toxicity of DON, the screening strategy based on toxicity assessment using Lemna minor was employed in the initial selection process. After incubation with DON, the culture filtrate from soil sample S5 showed no inhibitory effect on the growth of L. minor, while most of the other samples showed strong toxicity to L. minor similar to that of DON (Fig. 1a). Further analysis using GC-MS indicated that DON was either not degraded or converted into slightly less toxic metabolites, such as 3-keto-DON, in these culture filtrates that retained toxicity to L. minor (Fig. 1b and Supplementary Fig. 1). Notably, no DON or DON-derived metabolites were detected in the culture filtrate of sample S5, suggesting a complete degradation of DON (Fig. 1b). Moreover, a time-dependent detection by HPLC demonstrated that 3-epi-DON served as the intermediate product during DON degradation by consortium S5 (Supplementary Fig. 2). To isolate single colonies capable of completely degrading DON from S5 subcultures, the subcultures were treated with antibiotics. 16S rRNA high-throughput sequencing revealed that treatment with the antibiotic Penicillin G (PenG) enhanced the relative abundance of the Nocardioides genus. In contrast, this genus was absent in Erythromycin-treated subcultures, which lacked DON-degrading activity, indicating the potential role of the Nocardioides genus in DON degradation (Fig. 1c). Eventually, a bacterial strain (termed S5-5) that showed similar DON-degrading activity to the S5 consortium was obtained from the PenG-treated subculture (Supplementary Fig. 3). Based on morphological and phylogenetic analysis, it was identified as Nocardioides sp. (Fig. 1d and Supplementary Fig. 4).

The growth curve of strain S5-5 in a mineral medium containing DON at concentrations ranging from 10 to 1,000 μM demonstrated that strain S5-5 was capable of utilizing DON as the sole carbon and energy source (Fig. 1e). Additionally, strain S5-5 showed high degradation efficiency for DON in the culture medium and agricultural matrix under diverse conditions (Supplementary Fig. 5). Besides, it can also degrade multiple type A and B trichothecene mycotoxins that often co-contaminate grains with DON, such as Neosolaniol, 3-Acetyl deoxynivalenol, Nivalenol, and Fusarenon-X (Supplementary Fig. 6). To comprehensively explore the detailed process of DON complete degradation by S5-5, we meticulously carried out a time-dependent mass spectrometry assay targeting metabolites. Intriguingly, this assay unveiled that, in addition to 3-epi-DON, three hitherto-unknown intermediate metabolites (designated as M1, M2, and M3) emerged during the DON degradation process by S5-5 (Fig. 1f). These metabolites could not be detected using the HPLC-UV technique. Moreover, the increase of metabolites was disproportionate to the decrease of DON in relative abundance, and all of them disappeared at 24 hai (hours after inoculation) (Fig. 1g). Compared to the other three metabolites, M3 was the latest to reach its highest relative abundance (Fig. 1g). The further assay using these metabolites as substrates for strain S5-5 indicated that M3 was actually the catalytic product of 3-epi-DON. (Supplementary Fig. 7).

Notably, GC-MS profiles showed that the corresponding TMSI (N - (trimethylsilyl)imidazole) derivatives of M1, M2, and M3 have the same molecular ion at m/z 586.3 (Fig. 1h), leaving a 74-dalton (Da) difference compared to derivatized DON and 3-epi-DON (512.3). The displacement of a hydroxyl hydrogen atom with a trimethylsilyl group increases the molecular mass by 72 Da. This suggests that the formation of M1 and M2 from DON, and M3 from 3-epi-DON, involves the reduction of a ketone group to a hydroxyl group—leading to a 2 Da mass increase (Fig. 1h). We further confirmed this 2 Da mass difference between the three intermediates and their respective parent compounds via liquid chromatography-high resolution mass spectrometry (LC-HRMS) (Supplementary Fig. 8). Since DON and 3-epi-DON each contain only one ketone group, located at the C-8 position, the ketone reduction reaction must occur at C-8. To further elucidate the chemical structure, nuclear magnetic resonance (NMR) analysis was performed. Compared to the ¹H-NMR spectrum of DON, the spectrum of 8-OH-DON showed a new proton signal at approximately δ 4.27 ppm (a region characteristic of CH-OH groups). Additionally, significant chemical shift perturbations and multiplicity changes were observed for protons at C7 and C10 (H-7, H-10) (Supplementary Fig. 9; Table 1). These spectral changes are consistent with the expected electronic and structural effects of C8 ketone reduction. Thus, M1 and M2 are a pair of epimers, designated α- and β-8-hydroxyl-DON (8-OH-DON), respectively. Likewise, M3 is designated 8-hydroxyl-3-epi-DON (8-OH-3-epi-DON). These intermediate metabolites were further degraded by S5-5, and no signal typical of trichothecenes was detected by LC-HRMS under full scan mode at 24 hai (Supplementary Fig. 10a), while the culture filtrates from S5-5 post-DON degradation had no toxic effect on L. minor (Supplementary Fig. 10b, c). Moreover, obvious production of CO and 13CO was observed after degradation of DON by strain S5-5, where DON and stable carbon isotope (13C)-labelled DON were used as the sole carbon sources in MM medium (Supplementary Fig. 11). Taken together, these data indicated that DON was completely degraded and assimilated, serving as carbon sources for the growth of strain S5-5. Notably, this process was initiated by two distinct metabolic pathways: C3-epimerization and C8-reduction.

To identify candidate genes responsible for DON degradation, the genome of strain S5-5 was sequenced (GenBank accession No. CP199913), and a BAC library of strain S5-5 consisting of 1536 clones with an average insert size of about 140 kb was screened based on the enzymatic activity assay (Fig. 2a and Supplementary Figs. 12, 13). Paired-end sequencing of BAC clones and alignment to the reference genome of S5-5 revealed that thirteen genes were commonly shared among the inserts of 25 clones with 3-epi-DON forming activity (Supplementary Fig. 14), and eleven genes were commonly shared among the inserts of 29 clones with 8-OH-DON forming activity (Supplementary Fig. 15). A total of 24 genes were isolated from the strain S5-5 and expressed in E. coli. Among them, two recombinant proteins exhibited stable DON catalytic activity. One protein encoded by gene0108 (GenBank accession No. PX396128) catalyzes C3-epimerization of DON and is designated as DON 3R-epimerase (DONepi) (Fig. 2b, e, and Supplementary Fig. 16a). Kinetic analysis revealed that the kcat, Km, and kcat/Km of DONepi are 0.33 ± 0.04 min-1, 2.71 ± 0.85 mM, and 121.77 min-1 M-1, respectively (Supplementary Fig. 16b). Another protein encoded by gene4454 (GenBank accession No. PX396129) catalyzes C8-reduction of DON and is designated as DON 8-reductase (DONrd) (Fig. 2c, f, and Supplementary Fig. 17a). Kinetic analysis revealed that the kcat, Km, and kcat/Km of DONrd are 32.07 ± 2.84 min-1, 153.38 ± 29.06 μM, and 209.12 min-1 mM-1, respectively (Supplementary Fig. 17b). DONrd also contributes to the reduction of 3-epi-DON to produce 8-OH-3-epi-DON (Fig. 2d, g). Various chemical and metal ions affect the enzymatic activity of DONepi and DONrd to varying degrees (Supplementary Fig. 18).

DONepi and DONrd, which share a 25% amino acid identity, are most closely related to families AKR12 and AKR15, respectively (Supplementary Fig. 19). However, both of them have an identity of less than 40% relative to the members of these families. This suggests that they belong to new AKR families. Furthermore, a sequence similarity network (SSN) showed that most AKRs from both prokaryotes and eukaryotes were grouped into a primary cluster. This cluster included all AKRs from strain S5-5, with the exception of DONepi and DONrd, as well as other AKRs known for their activities in DON oxidation and 3-keto-DON reduction. (Fig. 2h). In contrast, DONrd was grouped into an additional smaller cluster, while DONepi had not been mapped to any cluster (Fig. 2h), which revealed the functional divergence of DONepi and DONrd.

Interestingly, the homologous proteins with the highest identity to these two specialized AKRs across all kingdoms of life were found in the genera Rhodococcus and Agromyces, sharing 75% and 55% amino acid identities with DONepi and DONrd, respectively. Based on the phylogenetic analysis, DONepi and its close homologs from the genera Nocardia, Rhodococcus, and Pseudonocardia were clustered together and distributed in a completely distinct branch separate from the Nocardioides clade, which is composed of nearly all homologs of DONepi from the Nocardioides genus (Supplementary Fig. 20). Similarly, DONrd was also clustered together with its close homologs from the genera Microbacterium, Agromyces, and Arthrobacter in an independent branch distinct from the clade composed of DONrd homologs from the Nocardioides genus (Supplementary Fig. 21). Moreover, synteny analysis revealed that the genomic sequences flanking the DONepi and DONrd genes respectively constitute a microsyntenic region that is conserved among bacterial species within the DONepi and DONrd clades. (Supplementary Figs. 20b and 21b). Thus, the occurrence of the DONepi and DONrd genes in the genome of strain S5-5 could be caused by horizontal gene transfer (HGT) events. Further analysis indicated that the DONepi and DONrd genes were located in two genomic islands (GIs) of the strain S5-5 genome, where the GC content was significantly different compared to the adjacent region of the genome. The predicted GIs including DONepi and DONrd have a large size of 13.2 kb and 18.9 kb, and contain fourteen and nineteen genes respectively, which, according to the COG annotation, are mainly related to metabolism (Fig. 2i, Supplementary Fig. 22, and Supplementary Tables 2, 3). Additionally, the microsyntenic regions related to DONepi and DONrd in the genomes of other bacteria from the DONepi and DONrd clades were also located in the GIs (Supplementary Figs. 23-26). In conclusion, these results revealed that DONepi and DONrd, acquired via GIs-mediated HGT, initiate the degradation of DON in two distinct metabolic pathways by epimerizing DON at C-3 position and reducing DON at C-8 position.

To determine the structure of DONepi, singleparticle cryo-EM was performed. Micrographs showed that particles with diverse angular coverage and secondary structural features could be observed in two-dimensional (2D) class averages (Supplementary Fig. 27). Further data processing resulted in a 3D reconstruction of DONepi with the dihedral symmetry (D4) and an overall resolution of 2.56 Å (Supplementary Fig. 27 and Table 4). DONepi assembles into an octameric structure (115×82 Å) (PDB ID: 9WK1). It is composed of two tightly-packed homotetramers with a 30° offset (Fig. 3a and 3b). Each protomer of DONepi contains a typical AKR signature structure-(α/β)8-barrel, and one coenzyme NADP+, which takes on an extended conformation. The nicotinamide ring on NADP+ is π - π stacked with Trp207, and the carboxamide group interacts with Thr155. Moreover, the adenine ring on NADP⁺ makes contact with Ser216 and Gln291 (Supplementary Fig. 28). The C-terminal tail loops of four protomers within one homotetramer act as anchors and mutually insert into the corresponding protomers in the other homotetramer, consequently forming an octameric quaternary structure (Fig. 3b). Although DONepi adopts a similar octameric architecture, the insert direction of anchors in DONepi is obviously different from that of other known AKRs in the family (Supplementary Fig. 29).

Regrettably, despite multiple attempts, we have not been able to obtain protein structure data with bound substrates, which may be attributed to weak binding affinity between the protein and substrate, among other potential factors. To examine the proposed mechanisms of DONepi catalysis, molecular docking analysis was conducted to characterize the putative interaction between the enzyme and substrate. Twenty interaction models of DONepi and DON with variances in their energies were predicted, and the model with a reasonable binding orientation, a lower binding energy and a larger number of hydrogen bonds was selected (Supplementary Table 5). DON, acting as a ligand, was located in the predicted active pocket of DONepi, and interacted with three DONepi residues via hydrogen bonds. Specifically, Tyr 27 (Y27) bound to the O8 and C7-OH of DON, Arg 86 (R86) to the C12,13-epoxy group, and His 124 (H124) to the C3-OH (Fig. 3c). Sequence alignment indicated that the residues Y27 and R86 represent a very rare amino-acid-substitution variation. Only 0.8% and 0.5% of the sequences in the top 1000 DONepi homologs possess such substitutions at these two positions (Fig. 3d). Moreover, most of the substitutions with Y and R at these two positions are distributed among the homologs in the DONepi clade (Fig. 3d). Two homologs with these specific substitutions from Rhodococcus (WP283336260) and Nocardia (WP063010560) exhibited similar DON-epimerizing activity to DONepi, while the homologs with one substitution of Tyr or without these specific substitutions, from Pseudonocardia (WP269804706) and Cellulomonas (WP291810687), showed very weak epimerization activity towards DON (Fig. 3e and Supplementary Fig. 30). We further performed reciprocal mutations between the DONepi and its homologs lacking DON-epimerizing activity. Replacing residue Y27 in DONepi with the corresponding residue W, or replacing residue R86 with H and G resulted in a significant reduction of DON-epimerizing activity (Fig. 3e). Additionally, H124 is the key residue making up the typical catalytic tetrad of AKRs coupled with D51, Y56, and K83, universally conserved among species (Fig. 3d). The mutation of each of them in DONepi could lead to the loss of DON-epimerizing activity (Fig. 3e), suggesting that the catalytic tetrad plays a crucial role in forming the active center of DONepi.

To investigate in depth the epimerization process, we analyzed the catalytic conversion of DON by DONepi using multiscale simulation methods, including molecular dynamics (MD), quantum mechanics/molecular mechanics (QM/MM) MD, and QM/MM well-tempered metadynamics (WT-MetaD). In a 600-nanosecond MD simulation of the enzyme-ligand complex, it was observed that the C3 hydrogen atom of DON consistently remained close to the nicotinamide C4' atom of the cofactor NADP+, with an average distance of approximately 2.5 Å (Supplementary Fig. 31). Additionally, the C3-OH group of DON formed a stable hydrogen bond with the phenolic hydroxyl group of the residue Y56 (H3···OTyr = 1.9 Å) (Supplementary Fig. 31). The proposed function of the conserved Y56 is to act as a general base in oxidation, assisting proton transfer and thereby promoting hydride transfer27,28. A snapshot of a dynamically equilibrated structure from the MD simulation was selected for subsequent QM/MM MD and WT-MetaD simulations to explore the catalytic mechanism in detail. The QM region included the entire DON molecule, part of the NADP+ molecule, and residue Y56, totaling 83 QM atoms. To describe the proton transfer (CV1) and hydride abstraction (CV2) involved in the oxidation process, two collective variables were defined: CV1 as the distance O3···H3 minus Y56O···H3, and CV2 as the distance C3···H minus C4···H (Fig. 4a). The evolution of the collective variables (CV1 and CV2) is shown in Supplementary Fig. 32. The results in Fig. 4b, c show that the reaction efficiently evolved from the initial DONepi-DON-NADP+ complex (denoted as MC) to the enzyme complex containing the 3-keto intermediate and NADPH (denoted as I). The free energy minima of the MC and I states were nearly isoenergetic, and separated by a unique intermediate M0 and a transition state TS. M0 is an intermediate formed after proton transfer from MC, with a free energy difference of less than 2 kcal/mol between them, indicating that proton transfer occurs relatively easily. Subsequently, the hydride ion transferred to the C4' atom of NADP+ via the transition state TS, forming the key 3-keto intermediate (see the Supplementary Movie 1). These results suggest that C3-epimerization of DON needs the oxidation of DON to form 3-keto-DON firstly, which proceeds through a concerted but asynchronous mechanism, where the activation energy required for hydride transfer determines the overall catalytic step (MC→TS) with a free energy barrier of 11.8 kcal/mol. Actually, DONepi can utilize 3-keto-DON as a substrate to form 3-epi-DON (Supplementary Fig. 33). However, 3-keto-DON was not detected during the transformation of DON to 3-epi-DON by DONepi, suggesting that it is a transient intermediate. To test whether 3-keto-DON is released from the enzyme active site during catalysis, hydroxylamine hydrochloride (NH2OH·HCl)—a strong nucleophilic reagent—was used as a competitive probe to target the carbonyl group of 3-keto-DON. We found that in the DONepi-catalyzed reaction system using 3-keto-DON as the substrate, hydroxylamine can readily outcompete NADPH, leading to almost complete oximation of free 3-keto-DON without enzymatic epimerization. In contrast, when hydroxylamine was added to the DONepi-catalyzed reaction system using DON as the substrate, the product detected was still 3-epi-DON, which is consistent with the reaction system without hydroxylamine (Supplementary Fig. 34). These data demonstrate that the intermediate 3-keto-DON is not released from the active site during the DON epimerization process catalyzed by DONepi. Furthermore, QM/MM MD simulations based on post-oxidation state conformation (denoted as I) revealed a rigid-body rotation of 3-keto-DON in the active site (see the Supplementary Movie 2). To simplify the description of this rotation, we defined a dihedral angle collective variable (CV3), constructed using the phenolic hydroxyl hydrogen atom of Tyr56, the carbonyl C3/O3 atoms, and the adjacent carbon C2 (Fig. 4d). The QM region included the 3-keto intermediate, the nicotinamide group of NADPH, and protonated Tyr56 (a total of 83 atoms). Three independent 5 ps unbiased QM/MM MD simulations consistently demonstrated that the 3-keto intermediate could spontaneously complete the rotation (Fig. 4e). The free energy profile constructed from the CV3 distribution revealed a rotational energy barrier ≤ 2.5 kcal/mol (Fig. 4f), confirming that this process is highly feasible under reaction conditions. Furthermore, this low-energy rotation provides the necessary conformational pre-organization for the subsequent NADPH-mediated reduction and epimerization, ensuring precise spatial orientation of the reactants. Overall, the octameric-structured DONepi catalyzes C3-epimerization of DON by directing the rotation of transient 3-keto intermediate to enable stereoinverting reduction of the keto group by NADPH in the active site.

Additionally, we generated transgenic Arabidopsis thaliana lines harboring the codon-optimized DONepi sequence and evaluated their DON response via seedling growth assays on medium supplemented with 5, 10, or 20 μM DON (Supplementary Figs. 35, 36). Root growth was comparable between transgenic and non-transgenic (wild-type) plants in DON-free medium. However, under all tested DON concentrations, transgenic lines exhibited longer roots and reduced leaf chlorosis (Supplementary Fig. 36). Specifically, at 20 μM DON, root lengths of lines DONepi-4 and DONepi-5 increased by 26% and 24% respectively, relative to non-transgenic plants (Supplementary Fig. 36). These results demonstrate that DONepi confers enhanced DON tolerance to transgenic A. thaliana.

DONrd catalyzed the reduction of DON at the C8 position to produce a pair of epimers, α- and β-8-OH-DON. These epimers could be caused by stereochemical hydride attacks from NADPH on the re-face and si-face of the prochiral C8 carbonyl (Fig. 5a). To examine the proposed mechanisms of DONrd catalysis, molecular docking analysis was conducted using the predicted structure by AlphaFold229 (Supplementary Fig. 37). DONrd also has a typical (α/β)8-barrel motif similar to DONepi but different C-terminal tail loop (Fig. 5b). Docking analysis revealed that DON acting as a ligand could bind into the predicted active pocket of DONrd via two binding orientations (Fig. 5c and Supplementary Table 6). One orientation is conducive to hydride attack at the si-face, in which DON C7-OH and C8 carbonyl group interacted with DONrd residues Y59 and H125 via hydrogen bonds, respectively (Fig. 5d ). Another orientation is conducive to hydride attack at the re-face, in which C8 carbonyl group, C12,13-epoxy group, and C15-OH of DON interacted with DONrd residues Y59, K129 and Y229 via hydrogen bonds, respectively (Fig. 5e). Among these, Y59 and H125 were the conserved residues making up the typical catalytic tetrad of AKRs coupled with D54, and K84 (Fig. 5f). The mutation of residue K84 in DONrd caused significant decrease in the reduction activity towards DON, and mutations in other three residues of the catalytic tetrad in DONrd could produce the loss of activity of DON reduction (Fig. 5g). Additionally, residues K129 and Y229 are also crucial for the reduction activity of DONrd. Replacing residue K129 in DONrd with residue H, the most frequently amino acid in the corresponding alignment position of residue K129, resulted in a significant reduction of DON-reducing activity (Fig. 5f, g). Interestingly, residue Y229 is conserved in these DONrd homologs and mutation could produce the loss of DON reduction activity (Fig. 5f, g). These data indicated that the reaction of reducing DON at C8 position catalyzed by DONrd is still dependent on the catalytic tetrad of D54-Y59-K84-H125, but residues K129 and Y229 paly the key role in allowing C8 carboxyl group to approach the catalytic tetrad in the appropriate orientation.

Moreover, using the C8-reduction products of DON as substrate for strain S5-5, we identified a intermediate metabolite designated M4 (Supplementary Fig. 38). The mass spectrum of TMSI-derivatized M4 showed a molecular weight of 674.5, an 88 Da increase compared to TMSI-derivatized 8-OH-DON (586.3). Since displacement of a hydroxyl hydrogen with a trimethylsilyl (TMSI) group increases molecular mass by 72 Da, this indicates an additional oxygen atom (16 Da) was incorporated into 8-OH-DON, introducing an extra hydroxyl group in M4. A previous study found that a cytochrome P450 enzyme mediates a similar catalytic process, hydroxylating DON at C16 to form 16-hydroxy-DON17. Notably, M4 was completely consumed with extended cultivation (Supplementary Fig. 38). These observations suggest that a potential oxidase (e.g., cytochrome P450 enzyme), along with other putative enzymes, contributes to the further degradation of the 8-hydroxylated intermediate, facilitating the complete degradation of DON (Fig. 6).

Methods
The aquatic plant Lemna minor was used for the toxicity assessment, which was grown in a half-strength Murashige and Skoog (1/2 MS) medium at 22 ℃ under a light regime of 16 h:8 h (light/dark). Arabidopsis thaliana (Col-0) was used for genetic transformation. Soil samples used for isolating DON-degrading bacteria were collected from wheat fields where Fusarium head blight is prevalent. Standard DON was purchased from Sigma-Aldrich (St. Louis, MO, USA). Stable carbon isotope (13C)-labelled DON was purchased from Alta Scientific Co., Ltd. (Tianjin, China) with catalog number 1ST7213C15-25A. 3-keto-DON and 3-epi-DON were prepared via biotransformation of DON, followed by isolation and purification using preparative high-performance liquid chromatography (preparative HPLC)32.

Soil samples were suspended in the sterile deionized water (w/v, 1:10), and supernatants after sedimentation were inoculated to MM medium (v/v, 1:10) with DON (100 μg mL-1). Following incubation at 28 °C with shaking at 220 rpm for 7 days, these cultures were filtered through a 0.22 μm filter membrane and used for toxicity tests with L. minor bioassay in a 48-well plate following the methods previously described with few modifications50. Sterilized culture filtrates starting from 100 μg mL-1 DON were diluted 10 times, resulting in an estimated final concentration of 10 μg mL-1 based on the amended DON-concentration. Three fronds of actively growing L. minor were placed in each well containing 0.8 mL diluted culture filtrates. The plants were incubated in triplicate for 6 days at 22 ℃ under a light regime of 16 h : 8 h (light/dark). The toxicity of DON-derived metabolites was analyzed based on the frond growth of L. minor. Culture samples with no significant inhibitory effects on L. minor growth were selected for isolating DON-degrading strains. The samples were treated with antibiotics to reduce microbial population diversity while maintaining community-level DON-degrading activity, and 16S rRNA gene sequencing was used to analyze the antibiotic-treated consortium for characterizing microbial population dynamics11,32.

High performance liquid chromatography (HPLC) analysis of the levels of DON and its metabolized products in culture samples was performed using an Agilent 1260 infinity series LC (Agilent, Santa Clara, CA, USA). Chromatographic separation was achieved using an Agilent Zorbax Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm) at 30 °C with methanol–water as mobile phases under gradient elution: initiation at 25% methanol; ramp to 75% over 15 min and then ramp to 80% over 5 min, hold for 3 min, then decrease back to 25% over 3 min, and hold for 3 min. The detection wavelength was 218 nm32.

Gas chromatography-mass spectrometry (GC-MS) analysis of trichothecene mycotoxins and their metabolites, derivatized by the mixture of trimethylsilyl imidazole/ trimethylchlorosilane (100:1 v/v), was performed using an Agilent 7890A GC equipped with an Agilent 5975C MS detector, and a DB-5MS capillary column (30 m × 0.25 mm × 0.1 µm, J&W Scientific, Santa Clara, CA, USA). High purity helium was used as the carrier gas with a flow rate of 1 mL/min. The GC oven temperature was initially held at 80 °C for 1 min, then increased linearly to 280 °C at a rate of 25 °C/min, and finally held at this temperature for 6 min. MS data were collected in full scan mode at mass range m/z 100 to 70025.

Ultrahigh-speed liquid phase tandem mass spectrometer (UPLC-HRMS/MS) analysis of putative DON metabolites was performed using a Thermo Scientific Accela 1250 UPLC (Thermo Fisher, Waltham, MA, USA) equipped with an AB SCIEX TripleTOF 6600 high-resolution mass spectrometer (SCIEX, Framingham, MA, USA). Chromatographic separation was achieved using a BEH Amide C18 column (2.1 m×100 mm, 1.7 μm) (Waters Co., Milford, MA, USA) at 30 °C with 5 mM ammonium acetate and methanol as mobile phases under gradient elution: initiation at 15% methanol, hold for 1 min; ramp to 90% methanol over 4.5 min, hold for 3 min, then decrease back to 15% over 1 min, hold for 1 min. The mass spectrometry acquisition signal mode was negative ion mode. The parameters of the mass spectrometry: capillary temperature, 320 °C; sprayvoltage, 3.00 kV; auxiliary gas heater temperature, 300 °C; and the full scan mass range was m/z 50-1000 Da51.

1H NMR spectra of DON and its metabolite were obtained at 298K using a Bruker Avance III 600 MHz spectrometer equipped with a Bruker inverse cryogenic probe (Bruker Biospin, Germany). The samples were dissolved in deuterated chloroform. 1H NMR chemical shifts were measured with relative to the residual chloroform signal and referenced to sodium 3-trimethylsilyl [2,2,3,3-d4] propionate (TSP-d4)32.

Strain S5-5 was cultured in nutrient broth (NB). Cells were harvested when the OD600 value reached 0.6-0.8, washed three times with sterilized water, and then inoculated into 5 mL of minimal medium (MM) in a 100 mL flask. The final cell concentration was adjusted to OD600 = 1, and the medium was supplemented with either 200 μM DON or a mixture of DON and ¹³C-labelled DON (molar ratio 50:1). The flask was incubated at 28 °C. After 7 days of incubation, the residual DON in the medium was detected by HPLC as described above. Gas in the flask was collected. The concentration of CO2 was analyzed using an Agilent 7890A GC equipped with a flame ionization detector (FID) and a HayeSep Q 80/100 UltiMetal column (12 ft L × 1/8″ OD × 2 mm ID, J&W Scientific, Santa Clara, CA, USA). High-purity nitrogen (N2) was used as the carrier gas. The FID temperature was set to 250 °C, with a hydrogen (H2) flow rate of 70 mL/min, a zero-grade air flow rate of 350 mL/min, and high-purity N2 as the makeup gas at 3.7 mL/min. The column oven temperature was maintained at 60 °C for 10 min. The δ13C value of 13CO2 was determined using a Trace Gas concentrator (Elementar Analysensysteme GmbH, Hanau, Germany) coupled to isotopic ratio mass spectrometer (IRMS) (IsoPrime100, GV Instruments, Manchester, UK). The collected gas was manually injected into the Trace Gas concentrator, then transported to the IRMS via helium carrier gas at a flow rate of 200 mL min-1. Ionized CO2 species were quantified as ionic currents using a multicollector system equipped with three Faraday cups. Ionic current intensities corresponding to mass-to-charge ratios 46, 45, and 44 were utilized to calculate the isotopic ratio R(13C/12C). This ratio was calibrated against the VPDB scale using a CO2 reference gas and expressed in parts per thousand (‰): δ13C = {[R(13C/12C)sample / R(13C/12C)standard] - 1} × 1000.

The genome of strain S5-5 was sequenced using a combination of Illumina and PacBio RS II single molecule real time (SMRT) sequencing platforms by Majorbio Biotech (Shanghai, China). The complete genome sequence was assembled using both the PacBio reads and Illumina reads, according to their standard procedures. The CDS prediction was achieved using Glimmer52. Functional annotation for the predicted protein-coding gene sequences was performed by alignment against public databases including COG, GO, KEGG, NR and SwissProt, using BLASTp with an e-value of 10−5.

A bacterial artificial chromosome (BAC) library for strain S5-5 was constructed according to Luo and Wing53 with some modifications. The high-copy composite vector, pHZAUBACFXJ1, was used for the BAC library construction. The restriction enzyme, BamH I, was employed to linearize the vector and digest the genomic DNA isolated from strain S5-5. Individual BAC clones were arrayed in 384-well plates and stored at −80 °C. To screen the BAC clone with DON degradation activity, 1 mL overnight culture for each BAC clone was suspended in 0.2 mL MM medium containing DON (30 μg mL-1). After incubation for 5 days at 37 °C, metabolites were extracted and analyzed by HPLC and GC-MS as described above. The BAC plasmid DNA was isolated from positive clones and sequenced at both ends. BAC end sequences were used to search for the gene information in the insert fragment by alignment against the genome sequence of strain S5-5 with BLASTn.

The selected genes were amplified by PCR using the genomic DNA of strain S5-5 as a template. The PCR products were cloned into the expression vector pET-28a (Novagen, Cat. No. 69337-3), and then transformed into Escherichia coli BL21 (DE3) for expression of the 6×His-tagged recombinant protein induced by isopropyl-β-D-thioga-lactopyranoside (IPTG). Recombinant proteins were purified by using Ni-NTA agarose (QIAGEN, Hilden, Germany) and analyzed by SDS-PAGE.

Enzymatic activity assays were performed in a 50 μL solution containing purified protein (6 μg), DON or its intermediate metabolites (100 μM), and either NADP⁺ (1 mM) or NADPH (0.25 mM), at different temperatures. The effect of pH on enzymatic activity was assessed at a constant temperature of 37°C using buffers (50 mM) with varying pH values: sodium acetate buffer (pH 4.0-5.8), sodium phosphate buffer (pH 5.8-7.5), Tris-HCl buffer (pH 7.5-8.6), and glycine-NaOH buffer (pH 8.6-10.0). The effects of temperature on enzymatic activity were tested for DONepi and DONrd in sodium phosphate buffer at pH 7.5 and pH 6.0, respectively. Unless otherwise specified, the standard enzyme activity assays for DONepi and DONrd were performed in sodium phosphate buffer (pH 7.5) at 45°C and sodium phosphate buffer (pH 6.0) at 25°C, respectively. Reactions were terminated by adding an equal volume of methanol. Substrates and their metabolites in the reaction solutions were detected by HPLC and GC-MS as described above. Reaction velocity was measured based on the reduction of the substrate. Origin 8 (Origin Software, Northampton, MA, USA) was used to calculate the Michaelis-Menten constants.

The protein sequences of about 370,000 AKRs in the InterPro database were downloaded. The SSN was then constructed via the EFI-EST web tool54 (http://efi.igb.illinois.edu/efi-est/) using 28297 UniRef50 cluster ID sequences from these AKRs as the input. Networks were generated with default parameters under an alignment score threshold of 45. The resulting representative node networks were visualized with Cytoscape v.3.755.

The large-scale purification of DONepi was performed using nickel-affinity chromatography (Qiagen). The protein was further purified successively by using ion exchange chromatography (Source 15Q 10/100, GE Healthcare) and size exclusion chromatography (Superdex 200 increase 10/300GL, GE Healthcare) in buffer (25 mM Tris, pH 7.5, 150 mM NaCl) on the AKTA pure system. For cryo-EM sample preparation, 3.5 μL of 1.0 mg mL-1 DONepi protein was dropped onto glow-discharged 300 mesh holey carbon grids (Quantifoil Au R1.2/1.3), which then were blotted using vitrobot Mark IV (ThemoFisher Scientific) with a blotting time of 3.5 sec at 100% humidity and 8 °C, and rapidly plunged into the liquid nitrogen pre-cooled liquid ethane. The cryo-EM data for DONepi was collected on a Titan Krios electron microscope operated at 300 kV, and equipped with a GatanK3 Summit detector and a GIF Quantum energy filter. Images for the DONepi protein were recorded in counting mode at 81,000× magnification, yielding a pixel size of 1.07 Å. Each image underwent dose fractionation into 32 frames, accumulating a total electron dose of 50 e⁻ Å⁻² during an exposure time of 3.5 s. A summit direct electron detector, featuring a 20 - eV slit width on its energy filter, was utilized. The defocus was preset within the range of -1.2 to -2.2 µm. For fully automated data collection, EPU software was employed. All images were motion-corrected by MotionCor256. A binning factor of 2 was applied along with concurrent dose weighting during this process. Subsequently, Gctf was utilized to estimate the defocus values57.

The cryo-EM dataset was processed in cryoSPARC (3.2)58. A total of 1,441,738 particles were automatically picked from 3,392 manually selected micrographs. After 2D classification, 1,127,418 good particles were selected and subjected to underwent multiple cycles of 3D classification. The 404,379 particles belonging to the best-scoring class were selected, followed by non-uniform and local refinement. The DONepi achieved a particle density with an estimated resolution of 2.56 Å according to the gold-standard Fourier shell correlation (FSC)59.

For the structure building of DONepi, the initial template was generated using AlphaFold. The initially fitted model was further refined through iterative model adjustments in COOT60. The resultant model was then refined against the map in real-space using PHENIX, with secondary structure and geometry restraints applied respectively61. Model quality was assessed via Molprobity scores and Ramachandran plots.

For molecular modeling and substrate docking, the three-dimensional structure of DONrd was predicted by AlphaFold2 online server with default parameters29. The coenzyme molecules, NADP+ or NADPH, were incorporated into the predicted models using AlphaFill online server (https://alphafill.eu/)62. Protein active pocket detection was achieved using Proteins Plus online server (https://proteins.plus/). Autodock 4.263 was used to perform the docking of modeled proteins and substrates with the genetic algorithm approach. The size of the grid box was set to 60 × 60 × 60 xyz points. Computational molecular docking was shown by Pymol software (Version 1.7.0.0, http://www. pymol.org/pymol).

Site-directed mutagenesis for the interacting amino acids was achieved using the Mut Express II Fast Mutagenesis (Vazyme, Nanjing, China) with recombinant plasmids pET28a-DONepi or pET28a-DONrd as the PCR template. A list of primers used for site-directed mutagenesis was shown in Supplementary Table 7. The sequences of mutant variants were confirmed by sequencing. The site-directed mutants of DONepi and DONrd were expressed, purified, and assayed for DON degradation activity following the same protocols described above.

The initial complex structure was derived from the Autodocking structure of DONepi enzyme in complex with DON and NADP⁺. To accurately assign the protonation states of titratable residues, we employed the Propka364, analyzing the local environment of each residue under the assumption of a system pH of 7.6. Based on the experimentally results and prior studies on related enzymes, the hydroxyl group of the residue Y56 was set to a deprotonated state. Subsequently, the enzyme-substrate complex was solvated in a cubic unit cell (dimensions: 100.168 × 100.095 × 99.880 Å³) using the solvate and genion modules of GROMACS 2023.5. The system contained 26,679 TIP3P water molecules and 10 Na⁺ ions to neutralize the overall charge. For classical molecular dynamics (MD) simulations, the following force field parameter sets were used: FF14SB for the protein, GAFF2 for the ligand DON and cofactor NADP⁺, and TIP3P for water molecules. To further enhance the accuracy of charge distribution, partial atomic charges for DON and NADP⁺ were calculated using Gaussian16 at the B3LYP/6-311++G** level of theory and fitted via the RESP method.

The MD simulations were performed using GROMACS 2023.5, following the protocol outlined below. First, the entire enzyme complex system underwent energy minimization. Next, weak positional restraints were applied to the protein, substrate, and cofactor in the NVT ensemble, and the system was simulated for 400 ps at 298 K to equilibrate the temperature. Subsequently, the system entered the density equilibration phase, where it was simulated for an additional 500 ps in the NPT ensemble. A time step of 2 fs was used, and all bonds involving hydrogen atoms were constrained using the LINCS algorithm. During the heating and density equilibration stages, weak restraints were applied to key reaction distances to maintain the substrate in its reactive position. The production phase simulations were carried out in the NPT ensemble, consisting of three independent 200 ns MD runs. Restraints were gradually released over the first 10 ns to allow the system to relax naturally. Supplementary Fig. 31 shows the changes in backbone RMSD during the simulation, indicating that the system achieved good stability.

To explore the reaction mechanism of the oxidation of the substrate DON to the key 3-keto intermediate, we performed a series of QM/MM (quantum mechanics/molecular mechanics) dynamic simulations using the CP2K 2024.1 program65. In the QM region, the dynamics of atoms depend on the electronic density ρ(r) calculated via density functional theory (DFT), while the dynamics of atoms in the MM region are governed by empirical force fields. In the QM/MM simulations, the QM part includes the amino acid residue Y56, the ligand DON, and the nicotinamide moiety and phosphate groups of NADP+, comprising a total of 83 atoms, enclosed in a cubic box with an edge length of 18.156 Å. The QM/MM boundaries (Cα and Cβ of NADP+ and Cα and Cβ of Tyr56) were treated using the link atom scheme, and the QM/MM interactions were computed using the GEEP scheme66. The QM part was described using DFT with the Perdew-Burke-Ernzerhof (PBE) functional67,68. All atoms were modeled using the DZVP-MOLOPT-GTH basis set, with a cutoff of 450 Ry retained for projecting the electronic structure. The parameters for the MM part were consistent with those used in classical MD simulations. Periodic boundary conditions were applied to both the QM and MM systems. Points of interest were extracted from the MD simulations, and geometries and velocities were retained to initiate QM/MM MD simulations in the NVT ensemble for 5 ps, using a Nose-Hoover thermostat with a time step of 0.5 fs.

To explore the free energy landscape (FEL) of the substrate oxidation reaction, we employed the well-tempered metadynamics (WT-MetaD)69 method at the same theoretical level as the QM/MM MD calculations. Two collective variables (CVs) were used, facilitated by the Plumed2 plugin (version 2.9.0). Each CV was defined as the difference between two distances that need to break or form during the reaction. The first CV measures the proton transfer between Y56 and the substrate 3-OH [CV1 = (O3···H3) - (OY56···H3)], while the second CV measures the interaction between the C3-H hydrogen atom of the substrate DON and the nicotinamide moiety of NADP+ [CV2 = (C3···H) - (C4···H)]. Thus, the second CV describes the hydrogen atom transfer between DON and nicotinamide. To enhance sampling in the CV space, a Gaussian kernel was added to the system every 50 MD steps (10 fs), with an initial height of 1.0 kcal mol-1 and a Gaussian width of 0.5 Å (corresponding to oscillations of CVs in unbiased dynamics, with a deposition interval of 10 fs). A bias factor γ = 15 was set to reduce the height of the generated Gaussian kernels. The time step for the QM/MM WT-MetaD simulation was adjusted to 0.2 fs, and the simulation was stopped upon meeting convergence criteria, with a total simulation time of 5 ps. Convergence was monitored using reweighting (debiasing) methods and block analysis, and errors in the free energy estimates were calculated.

The DONepi gene was codon-optimized for expression in Arabidopsis thaliana and synthesized (Supplementary Table 8). Subsequently, it was integrated into the vector pRI101-AN (Takara, Code No. 3262). The recombinant plasmid carrying the DONepi gene was transformed into the Agrobacterium tumefaciens strain GV3101, which was used to transformed Arabidopsis thaliana through the floral dip method. Positive transgenic plants were selected on half-strength Murashige and Skoog (MS) medium supplemented with 50 mg L-1 kanamycin and further verified by PCR.

For the DON tolerance assay, seeds of Arabidopsis thaliana were surface-sterilized by using 10% sodium hypochlorite, followed by incubation on the half-strength MS medium. seedlings with uniform shoot lengths (2-3 mm) were transferred to fresh half-strength MS medium supplemented with DON at final concentrations of 5, 10, and 20 μM, respectively. Seedlings were then cultured under a 16 h light/8 h dark photoperiod at 22-24 °C. For each experimental line, at least 15 independent seedlings were used, and their root lengths were measured 21 days post-transfer.

All assays were performed in triplicate. The Student’s t-test (unpaired, two-tailed) was used to evaluate the statistical differences between groups using the GraphPad Prism software. P < 0.05 was considered statistically significant. Asterisks indicate P values (* P < 0.05, ** P < 0.01).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript reports the identification of two enzymes from the bacterium Nocardioides sp. S5-5 that are able to transform DON, a mycotoxin produced by the fungus Fusarium that infects grain crops. DON therefore poses significant health risk to humans and livestock. Certain microbial enzymes can catalyze the transformation of DON to its 3-hydroxy epimer (3-epiDON) that is significantly less toxic. Previous studies showed that this epimerization reaction is normally carried out by two enzymes; A PQQ dependent enzyme that oxidizes DON to 3-ketoDON and an NADPH dependent aldo-ketoreductase that reduces 3-keto DON to 3-epiDON. The authors of this paper suggested that the bacterium Nocardioides sp. S5-5 utilized a single aldo-keto reductase enzyme (named DONepi) to transform DON to 3-epiDON through the intermediate 3-ketoDON. Structural determination of DONepi by Cryo-EM followed by molecular dynamics simulation of ligand interaction suggest that this could occur by rotation of the substrate in the active site relative to the catalytic residues and the nicotinamide cofactor, enabling the stereoselective two step transformation reaction. The authors also isolated another enzyme, DONrd, that was proposed to catalyze the reduction of the 8-keto group of DON and 3-epiDON to their 8 hydroxy derivatives. However, toxicity of the 8-hydroxy derivatives is not determined.\nThis is an interesting study that has potential significance for the biological detoxification of an important mycotoxin. However, there are some concerns that the authors should address to improve the manuscript.\n1. Some experimental evidence should be provided to support the premise from molecular dynamics simulation that DONepi catalyzes a two step reaction through an enzyme bound 3-ketoDON intermediate. For example (i) determine if DONepi also utilizes 3-ketoDON as a substrate. (ii) Determine the stoichiometry through spectrophotometric progress curve of NADPH utilization/formation when DON is transformed to 3-epiDON ? (iii) Provide time course data to confirm if 3-ketoDON can be detected, especially in the early phase of the enzyme catalyzed reaction.\n2. Related to point (1) above, the proposed mechanism would suggest that reaction catalyzed by DONepi would produce an equilibrium mixture of DON and 3-epiDON. The authors should comment on whether this was observed and the implications for the use of this enzyme for complete detoxification of DON.\n3. It is questionable that Nocardioides sp. S5-5 can completely degrade DON. There was minimal increase in OD600 when the bacteria were grown with DON as a sole carbon source (Fig 1e). In addition, the bacteria did not reach stationary phase after 144h (Fig 1e) although DON and 3-epiDON appeared to be completely transformed within 24 h (Fig 1g). Without additional experiments (eg by using radiolabelled DON) it is uncertain that the bacteria can transform DON to water and CO2 (Fig 6).\n4. Pgs 18 and 19. Description of characterization of transgenic plants is not complete. Codon optimized DONepi sequence should be provided in the supplementary information. Assays for DON tolerance should be described.\n5. Lines 304-306 and 310-312. Results from transgenic plants and growth on 8-OHDON should be presented in the results section instead of the discussion.\n6. Effects of DON on Arabidopsis is modest. Other than a slight reduction in root length, wild-type plants appeared healthy when exposed to DON which makes comparison with the transgenic plants difficult. It is likely that the concentration of DON used in the assay was too low (refer to Poppenberger J. Biol. Chem. 2003, 278, pp. 47905–47914 for comparison).\n7. Lines 310-312 + Suppl fig 35. “We found that the 8-hydroxyl intermediate produced by DONrd catalysis can be further degraded by a potential cytochrome P450 enzyme and other putative enzymes (Fig. 6 and Supplementary Fig. 35)”. What is the evidence that the enzyme responsible is a cytochrome P450 ?\n8. It is not clear how that authors can confidently assign the 8-OH proton peak and identify 8-OH DON as the compound produced from a 1D proton NMR spectrum (Suppl Fig 9).\n9. Pg 429-435 Method for enzyme activity measurements require more details. Eg. Is a time course performed. How are products quantified by HPLC or GC/MS ? What precautions are taken to ensure that initial velocity was measured. Also ,what is the buffer and temperature used for a standard assay eg data in Fig 3e, 5g, Suppl Fig 17 etc.\n10. Genome sequence from Nocardioides sp. S5-5 should be deposited in GENBANK and the accession numbers for the genome, DONepi and DONrd should also be provided.\n11. Cryo EM structure should be deposited in the PDB database and the PDB ID and validation report should be provided.\n13. Suppl Fig 20. Mistake in Figure legend. “DONepi” should be “DONrd”\n14. Suppl Fig19b and 20b. Provide Genbank accession or uniport ID of homologs of DONepi and DONrd", "reviewer_2": "Comments:\nIn this manuscript, the authors report a comprehensive study on the absolute degradation of DON. The work can be roughly divided into 3 parts:\n1. Identification of Nocardioides sp. S5-5 is capable of completely degrading DON through a mechanism involving C3-epimerization and C8-reduction.\n2. Discovery of two horizontally transferred aldo-keto reductases (AKR), DONepi and DONrd, for C3-epimerization and C8-reduction, respectively. This is the most interesting part of this work.\n3. Structural and mechanistic elucidation of DONepi and DONrd using cryo-EM and QM/MM simulations.\nOverall, this study presents a novel mechanism for DON degradation and identifies the key enzymes involved.\nHowever, I think this work is not suitable for publishing in Nature Communications, since there are several concerns, particularly regarding the novelty, practical applications of the strain and enzymes, as well as some mechanistic and structural limitations.\nMajor concerns:\n1. There are many enzymes similar to aldo-keto reductase DONepi that have been reported to degrade DON, targeting 3-OH. Additionally, a strategy to target the epoxy group and 3-keto using a multi-enzyme cascade reaction has also been reported (Hou B, https://doi.org/10.1016/j.foodchem.2024.139886). Therefore, it seems that the novelty of degrading DON using these enzymes is limited.\n2. Biosafety of the strain, it is essential to assess the biosafety of Nocardioides sp. S5-5 in industrial applications. Has the strain been screened for potential production of other harmful metabolites or toxins? Is there any genomic or metabolomic evidence supporting its safety for animal or environmental use?\n3. The authors emphasize the practical potential of these enzymes; however, these enzymes still require co-factor NADP or NADPH to initiate the degradation. Therefore, the authors did not adequately address the concerns they raised in the introduction (Lines 43-47), which remain unresolved in the current work.\n4. While the authors show that transgenic A. thaliana expressing DONepi and DONrd exhibit resistance to DON, this model system may lack translational relevance due to the following reasons: 1) FHB primarily affects wheat, using transgenic wheat would provide more substantial and more relevant evidence for agricultural applications; 2) DON toxicity is a significant concern in animals and humans via contaminated feed. Therefore, it would be more meaningful to evaluate the detoxification efficiency of these enzymes or the strain in animal models or to simulate feed detoxification conditions.\n5. The study focuses on two enzymes involved in the initial steps of DON degradation, but the complete pathway of “absolute degradation” is not fully established. Does Nocardioides sp further metabolize the products of DONepi and DONrd. S5-5?\n6. Although the authors utilize cryo-EM and QM/MM simulations to study the catalytic mechanisms, the absence of substrate- or product-bound structures significantly limits mechanistic insights. Given that both enzymes are soluble and expressed in E. coli, did the authors attempt to obtain X-ray structures with ligands? X-ray crystallography offers greater flexibility for soaking experiments and higher resolution, potentially facilitating the visualization of key interactions with substrates or products.\nOther minor concerns and suggestions for the authors to consider:\n1. The introduction provides a summary of DON-degrading enzymes, but several important and relevant studies are missing. The authors should consider citing: enzymatic degradation of T-2 toxin and DON (Yang J, https://doi.org/10.1021/acscatal.5c03462; Huang J, https://www.nature.com/articles/s41589-019-0446-8 ); An interesting and similar strategy to target epoxy group and 3-keto using a multi-enzyme cascade reaction (Hou B, https://doi.org/10.1016/j.foodchem.2024.139886);\n2. The manuscript lacks kinetic parameters for DONepi and DONrd. Determining parameters such as kcat, Km, and catalytic efficiency would significantly strengthen the characterization of these enzymes\n3. Line 95, “24 hai” appears to be a typographical error. Should this be “24 h”?\n4. In Lines 36–37, the authors state that 3-epi-DON has the lowest toxicity among DON metabolites based on a 2015 reference. However, recent studies targeting the epoxy group (a major contributor to DON toxicity) have shown promising detoxification outcomes. The authors should discuss whether 3-epi-DON remains less toxic than metabolites derived from the degradation of the epoxy group.\n5. In Supplementary Table 4 and Methods (Lines 466–469), the authors do not provide information on the initial model used for DONepi structure determination. Clarifying the template and method used for initial model building (e.g., AlphaFold prediction, homology modeling) is essential for reproducibility and transparency."}, "author_response": "A list of responses to reviewers\nWe thank the anonymous referees very much for assisting in the improvement of our\nmanuscript. We have made all necessary changes/responses based on the advice and\ncomments/suggestions made by the reviewers. All changes, as well as our responses,\nare outlined below as they appear in the order of the comments and suggestions.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript reports the identification of two enzymes from the bacterium\nNocardioides sp. S5-5 that are able to transform DON, a mycotoxin produced by the\nfungus Fusarium that infects grain crops. DON therefore poses significant health risk\nto humans and livestock. Certain microbial enzymes can catalyze the transformation of\nDON to its 3-hydroxy epimer (3-epiDON) that is significantly less toxic. Previous\nstudies showed that this epimerization reaction is normally carried out by two enzymes;\nA PQQ dependent enzyme that oxidizes DON to 3-ketoDON and an NADPH dependent\naldo-ketoreductase that reduces 3-keto DON to 3-epiDON. The authors of this paper\nsuggested that the bacterium Nocardioides sp. S5-5 utilized a single aldo-keto reductase\nenzyme (named DONepi) to transform DON to 3-epiDON through the intermediate 3-\nketoDON. Structural determination of DONepi by Cryo-EM followed by molecular\ndynamics simulation of ligand interaction suggest that this could occur by rotation of\nthe substrate in the active site relative to the catalytic residues and the nicotinamide\ncofactor, enabling the stereoselective two step transformation reaction. The authors also\nisolated another enzyme, DONrd, that was proposed to catalyze the reduction of the 8-\nketo group of DON and 3-epiDON to their 8 hydroxy derivatives. However, toxicity of\nthe 8-hydroxy derivatives is not determined.\nThis is an interesting study that has potential significance for the biological\ndetoxification of an important mycotoxin. However, there are some concerns that the\nauthors should address to improve the manuscript.\n1. Some experimental evidence should be provided to support the premise from\nmolecular dynamics simulation that DONepi catalyzes a two-step reaction through an\nenzyme bound 3-ketoDON intermediate. For example (i) determine if DONepi also\nutilizes 3-ketoDON as a substrate. (ii) Determine the stoichiometry through\nspectrophotometric progress curve of NADPH utilization/formation when DON is\ntransformed to 3-epiDON?\nResponse (Lines 249-250; Supplementary Fig. 33): Thank you for the careful\nguidance about how to improve our manuscript. As per your suggestion, we have\ncarried out the new experiments, and the results showed that DONepi was able to utilize\n3-keto-DON as a substrate to form 3-epi-DON. To be more convincing and aligned with\nyour concerns, we have included a new figure (Supplementary Fig. 33) in the\nsupplementary materials related to the utilization of 3-keto-DON by DONepi as a\nsubstrate. More detailed interpretation was included in Lines 249-250: “Actually,\nDONepi can utilize 3-keto-DON as a substrate to form 3-epi-DON (Supplementary\nFig. 33).”\nSupplementary Fig. 33. Catalytical capacity of DONepi using 3-keto-DON as the\nsubstrate. 3-keto-DON and its catalyzed product by DONepi were detected using HPLC.\nIn addition, a time-course detection was performed to monitor the transformation of\nDON to 3-epi-DON, with NADP⁺ as the coenzyme. As shown in the figures below (Figs.\nA1, A2), no signals corresponding to 3-keto-DON or NADPH were detected in this\nreaction. This could be because the formed 3-keto intermediate is not released from the\nenzyme and is transiently reduced using NADPH as the coenzyme. A similar\nreaction process was uncovered in the C4-epimerization of UDP-Glucuronic Acid\n(UDP-GlcA) by UGAepi, in which a transient 4-keto intermediate that is not released\nfrom the enzyme was also utilized by UGAepi to achieve the epimerization (Borg et al.,\nFEBS J. 2021, 288: 1163-1178; Borg et al., Angew. Chem. Int. Ed. 2023, 62:\ne202211937).\nFigure A1. HPLC analysis of DON epimerization catalyzed by DONepi. Time-course\ndetection of DON and its metabolite was performed over 0-24 h post-incubation with DONepi.\nArrows indicate the intermediate product, 3-epi-DON.\nFigure A2. Spectrophotometric analysis of NADPH formation during DON\nepimerization by DONepi with NADP⁺ as the coenzyme. Time-course detection of the\noptical density at 340 nm (characteristic of NADPH) was performed over 0-24 h post-incubation\nwith DONepi. Values are presented as the means of three replicates; error bars represent standard\ndeviation.\n2. Related to point (1) above, the proposed mechanism would suggest that reaction\ncatalyzed by DONepi would produce an equilibrium mixture of DON and 3-epiDON.\nThe authors should comment on whether this was observed and the implications for the\nuse of this enzyme for complete detoxification of DON.\nResponse (Line 347-350; Line 356-359): Thank you for pointing this out. In light of\nthe current findings, while the standalone use of DONepi is capable of effectively\nreducing the concentration of DON, it does result in the formation of an equilibrium\nmixture of DON and 3-epi-DON. To achieve the complete detoxification of DON, the\nsynergistic action of DONepi and other subsequent enzymes—including those that\nutilize 3-epi-DON (the reaction product of DON catalyzed by DONepi) as their\nsubstrate—is indispensable (Line 347-350). As a feasible strategy, all enzymes\ninvolved in the complete degradation pathway of DON (including DONepi) can be co-\nexpressed in engineered strains (e.g., yeast, E. coli). This approach will facilitate the\ndevelopment of an efficient enzyme reagent for the complete degradation of DON in\nfuture applications (Line 356-359). We have now revised the Discussion section to\naddress these points.\n3. It is questionable that Nocardioides sp. S5-5 can completely degrade DON. There\nwas minimal increase in OD600 when the bacteria were grown with DON as a sole\ncarbon source (Fig 1e). In addition, the bacteria did not reach stationary phase after\n144h (Fig 1e) although DON and 3-epiDON appeared to be completely transformed\nwithin 24 h (Fig 1g). Without additional experiments (eg by using radiolabelled DON) it is uncertain that the bacteria can transform DON to water and CO2 (Fig 6).\nResponse (Lines121-124; Lines 448-469; Supplementary Fig. 11): We appreciate the\nreviewer's critical assessment of DON degradation completeness by Nocardioides sp.\nS5-5. In Figure 1e, strain S5-5 entered the stationary phase after 48-72 hours in MM\nmedium containing 100 μM DON. This delay relative to DON's disappearance (24\nhours, Figure 1g) is likely due to the sequential metabolic processes required for\ndegrading downstream intermediates beyond the initial DON transformation steps.\nFor the 1000 μM DON treatment, the extended growth phase (without reaching stationary\nphase by 144 hours) probably reflects the longer time required to completely degrade\nthe higher toxin concentration.\nTo further verify that strain S5-5 completely mineralizes DON to CO , we performed\n2\ntwo new experiments, which measured CO production and the δ13C values of respired\n2\ncarbon source in MM medium. The results showed that: (1) In MM medium with DON\nas the sole carbon source, a significant increase in CO levels was detected after strain\nDON was used as the sole carbon source, substantial 13CO production was observed\n2\nfollowing degradation by strain S5-5 (Supplementary Fig. 11d, e).\nThese results collectively confirm that DON is indeed completely degraded by strain\nS5-5 into CO . We have added a brief description of this conclusion in the revised\nMaterials and Methods section (Lines 448-469). We hope these explanations and new\nexperimental data effectively address the reviewer's concerns.\nSupplementary Fig. 11. Detection of CO production following absolute\n2\ndegradation of DON by strain S5-5. DON and 13C-labelled DON were used as the sole\ncarbon sources in MM medium. At 7 days post inoculation (dpi) with strain S5-5, the concentration\nof CO and the δ13C content of respired CO were detected by gas chromatography (GC) and isotope\n2 2\nratio mass spectrometry (IRMS), respectively. a, HPLC analysis of DON levels in the medium at 7\ndpi. b, GC analysis of CO production at 7 dpi. c, Average CO concentration in samples at 7 dpi.\n2 2\nd, Representative ionic current intensities of masses 46, 45, and 44 (upper, middle, and bottom\npanels, respectively) acquired from IRMS for each treatment. e, δ13C values of respired CO in\n2\nsamples at 7 dpi. Data are from three independent biological experiments.\n4. Pgs 18 and 19. Description of characterization of transgenic plants is not complete.\nCodon optimized DONepi sequence should be provided in the supplementary\ninformation. Assays for DON tolerance should be described.\nResponse (Supplementary Table 8; Lines 627–632): Thank you for the careful\nguidance. As per your suggestion, we have added the codon-optimized DONepi\nsequence to the Supplementary Information as Supplementary Table 8. Additionally,\nwe have included a paragraph in the Materials and Methods section to specify the\ndetails of the DON tolerance assays (Lines 627-632).\n5. Lines 304-306 and 310-312. Results from transgenic plants and growth on 8-\nOHDON should be presented in the results section instead of the discussion.\nResponse (Lines 266–274; Lines 300–310): We appreciate the valuable suggestion.\nWe have now included two new paragraphs presenting the results from transgenic plants\n(Lines 266–274) and the further degradation of 8-OH-DON (Lines 300–310) in the\nResults section.\n6. Effects of DON on Arabidopsis is modest. Other than a slight reduction in root length,\nwild-type plants appeared healthy when exposed to DON which makes comparison\nwith the transgenic plants difficult. It is likely that the concentration of DON used in\nthe assay was too low (refer to Poppenberger J. Biol. Chem. 2003, 278, pp. 47905–\n47914 for comparison).\nResponse (Lines 266–274): Thank you for your comments. We have indeed noted the\nDON concentrations (15-100 μM) used in the reference you mentioned, which is\nalready cited as ref. 16 in our original reference list. DON is known to preferentially\ninhibit root elongation, and 10 μM DON has been shown to severely inhibit root growth\n(Masuda et al., J. Exp. Bot., 2007, 58: 1617-1626). Similar DON concentrations (3-20\nμM) have also been used in recent DON resistance assays (Mandala et al., MPMI, 2019,\n32: 583-592; Wang et al., Nat. Commun., 2021, 12: 6426; He et al., Plant Biotech. J.,\n2024, 22: 2395-2409). Based on our experimental experience and a comprehensive\nreview of these references, we selected three DON concentrations (5, 10, and 20 μM)\nfor this study. Our results showed that, at all three tested concentrations, the root length\nof transgenic Arabidopsis thaliana harboring the DONepi gene was significantly longer\nthan that of wild-type plants (P < 0.01).\nWe apologize for the overly simplistic description of this section in the original\nmanuscript, which may have reduced readability—this was due to word count\nconstraints. We have now added a new paragraph as per your suggestion to clarify the\ndetailed root length differences between transgenic and wild-type plants grown on\nDON-containing medium (Lines 266–274). We agree that higher DON concentrations\nmight amplify the difference in DON resistance between transgenic and wild-type\nplants. However, we believe our current results are sufficient to support the conclusion\nthat the DONepi gene confers enhanced DON tolerance to transgenic A. thaliana.\n7. Lines 310-312 + Suppl fig 35. “We found that the 8-hydroxyl intermediate produced\nby DONrd catalysis can be further degraded by a potential cytochrome P450 enzyme\nand other putative enzymes (Fig. 6 and Supplementary Fig. 35)”. What is the evidence\nthat the enzyme responsible is a cytochrome P450?\nResponse (Lines 300–310): We appreciate the reviewer’s concern regarding the\nevidence supporting that a cytochrome P450 enzyme may be responsible for the further\ndegradation of the 8-hydroxyl intermediate produced by DONrd. This speculation is\nbased on our mass spectrometry data: when 8-OH-DON was used as a substrate for\nstrain S5-5, we detected an intermediate (designated M4), and GC-MS analysis\nindicated that an additional oxygen atom (16 Da) was incorporated into 8-OH-DON,\nintroducing an extra hydroxyl group in M4 (Supplementary Fig. 37). A previous study\ndemonstrated that a cytochrome P450 enzyme mediates a similar catalytic process,\nspecifically hydroxylating DON at C16 to form 16-hydroxy-DON (ref. 18). Based on\nthis analogy, we speculated that a related cytochrome P450 enzyme may be involved in\nthis catalysis.\nWe acknowledge that the original description of this part was overly simplistic. As\nsuggested, we have added a new paragraph in the Results section to clarify these results\nand the basis for our speculation (Lines 300–310).\n8. It is not clear how that authors can confidently assign the 8-OH proton peak and\nidentify 8-OH DON as the compound produced from a 1D proton NMR spectrum\n(Suppl Fig 9).\nResponse (Lines 105–116): Thank you for your insightful comment. We apologize for\nthe lack of detailed description of these results in the original manuscript, which may\nhave caused confusion during your review. Our identification of 8-OH-DON is not\nbased on ¹H-NMR alone but is instead supported by converging evidence from multiple\nanalytical techniques, which we will outline below. This multi-faceted approach allows\nus to make a confident assignment. To clarify our structural identification process, we\nhave added a detailed description in the Results section (Lines 105–116), which reads\nas follows: “This suggests that the formation of M1 and M2 from DON, and M3 from\n3-epi-DON, involves the reduction of a ketone group to a hydroxyl group—leading to\na 2 Da mass increase (Fig. 1h). We further confirmed this 2 Da mass difference between\nthe three intermediates and their respective parent compounds via liquid\nchromatography-high resolution mass spectrometry (LC-HRMS) (Supplementary Fig.\n8). Since DON and 3-epi-DON each contain only one ketone group (located at the C-8\nposition), the ketone reduction reaction must occur at C-8. To further elucidate the\nchemical structure, nuclear magnetic resonance (NMR) analysis was performed.\nCompared to the 1H-NMR spectrum of DON, the spectrum of 8-OH-DON showed a\nnew proton signal at approximately δ 4.27 ppm (a region characteristic of CH-OH\ngroups). Additionally, significant chemical shift perturbations and multiplicity changes\nwere observed for protons at C7 and C10 (H-7, H-10) (Supplementary Fig. 9; Table 1).\nThese spectral changes are consistent with the expected electronic and structural effects\nof C8 ketone reduction.”\n9. Pg 429-435 Method for enzyme activity measurements require more details. Eg. Is a\ntime course performed. How are products quantified by HPLC or GC/MS? What\nprecautions are taken to ensure that initial velocity was measured. Also, what is the\nbuffer and temperature used for a standard assay eg data in Fig 3e, 5g, Suppl Fig 17 etc.\nResponse (Lines 498-508): We appreciate the reviewer’s valuable comments regarding\nthe method for enzyme activity measurements. To address your concerns, we provide\ndetailed clarifications below:\n(1) Time course analysis: A time course experiment was routinely performed to monitor\nthe change in substrate concentration over the course of the reaction. This allowed\nus to confirm the linear phase of the reaction, which is critical for subsequent\nmeasurements.\n(2) Quantification approach: Due to the lack of commercial standards for the newly\nidentified products, we quantified the enzyme activity by measuring the reduction\nof the substrate (rather than direct product quantification) via HPLC, as referenced\nin the original text.\n(3) Ensuring initial velocity: To ensure initial reaction velocity was measured, we first\noptimized the enzyme concentration—specifically, we verified that the reaction\nvelocity remained linear with respect to both reaction time and enzyme amount\nunder our experimental conditions. This linearity confirms that measurements were\nconducted within the initial phase of the reaction, avoiding substrate depletion or\nproduct inhibition.\n(4) Standard assay conditions: We have now added detailed information about the\nstandard assay conditions (including buffer type, pH, and temperature) in the\nMethods section (Lines 498-508). For example, the assays corresponding to Fig.\n3e, 5g, and Supplementary Fig. 17 were performed in 50 mM sodium phosphate\nbuffer (pH 7.5 at 45°C for DONepi; pH 6.0 at 25°C for DONrd), which is consistent\nwith the optimized conditions described in the revised Methods.\nWe apologize for the insufficient details in the original manuscript and believe these\nclarifications, along with the revised Methods section, will address your concerns.\n10. Genome sequence from Nocardioides sp. S5-5 should be deposited in GENBANK\nand the accession numbers for the genome, DONepi and DONrd should also be\nprovided.\nResponse (Lines 129, 138, 142): Thank you for your valuable suggestion. We have\ndeposited the genome sequence of Nocardioides sp. S5-5, as well as the coding\nsequences of DONepi and DONrd, in the GenBank database of NCBI. Their respective\naccession numbers are CP199913, PX396128, and PX396129, which have been added\nto the revised manuscript.\n11. Cryo EM structure should be deposited in the PDB database and the PDB ID and\nvalidation report should be provided.\nResponse (Line 182; Supplementary Table 4): As per your suggestion, we have\ndeposited the cryo-EM structure in the PDB database. The corresponding PDB ID\n(9WK1) has been added to the Results section (Line 182) and Supplementary Table\n4 of the revised manuscript. Additionally, we have provided the validation report as\nadditional supplementary information.\n12. Suppl Fig 17. Indicate the anions of the salts eg. are they chloride salts?\nResponse (Supplementary Fig. 18): Thank you for your careful suggestion. We have\nnow specified the anions of the salts in the legend of Supplementary Fig. 18. Except\nfor Zn²⁺ and Cu²⁺ (which are sulfates), all other salts are chlorides.\n13. Suppl Fig 20. Mistake in Figure legend. “DONepi” should be “DONrd”\nResponse (Supplementary Fig. 21): Thank you for your careful check. We apologize\nfor this oversight. We have corrected the mistake in the legend of Supplementary Fig.\n21, replacing “DONepi” with “DONrd” as suggested.\n14. Suppl Fig19b and 20b. Provide Genbank accession or uniport ID of homologs of\nDONepi and DONrd\nResponse: Thank you for your comment. We appreciate your attention to the details of\nthe homologs. In fact, the GenBank accession numbers of the DONepi and DONrd\nhomologs have been provided in Supplementary Figs. 20b and 21b—specifically,\nthey are listed in the parentheses directly below each homolog’s gene name in the\nfigures. To ensure clarity, we have double-checked and confirmed the accuracy of these\naccession numbers in the revised figures. We apologize for any inconvenience caused\nby the potential oversight in locating this information, and we hope this clarification\nhelps you easily find the required details.\nReviewer #2 (Remarks to the Author):\nComments:\nIn this manuscript, the authors report a comprehensive study on the absolute\ndegradation of DON. The work can be roughly divided into 3 parts:\n1. Identification of Nocardioides sp. S5-5 is capable of completely degrading DON\nthrough a mechanism involving C3-epimerization and C8-reduction.\n2. Discovery of two horizontally transferred aldo-keto reductases (AKR), DONepi and\nDONrd, for C3-epimerization and C8-reduction, respectively. This is the most\ninteresting part of this work.\n3. Structural and mechanistic elucidation of DONepi and DONrd using cryo-EM and\nQM/MM simulations.\nOverall, this study presents a novel mechanism for DON degradation and identifies the\nkey enzymes involved.\nHowever, I think this work is not suitable for publishing in Nature Communications,\nsince there are several concerns, particularly regarding the novelty, practical\napplications of the strain and enzymes, as well as some mechanistic and structural\nlimitations.\nMajor concerns:\n1. There are many enzymes similar to aldo-keto reductase DONepi that have been\nreported to degrade DON, targeting 3-OH. Additionally, a strategy to target the epoxy\ngroup and 3-keto using a multi-enzyme cascade reaction has also been reported (Hou\nB, https://doi.org/10.1016/j.foodchem.2024.139886). Therefore, it seems that the\nnovelty of degrading DON using these enzymes is limited.\nResponse (Lines 329–332; Lines 342-343): Thank you for your critical comment,\nwhich has prompted us to clarify the key distinctions between our work and previously\nreported studies on DON-degrading enzymes—points we recognize may not have been\nsufficiently emphasized in the original manuscript.\nWe fully agree that aldo-keto reductases (AKRs) related to DON metabolism have been\nreported, but these enzymes differ fundamentally from the novel AKR (DONepi)\nidentified in our study in terms of both catalytic function and reaction mechanism.\nAs noted in our previous work and others (refs. 25, 30-32), known DON-associated\nAKRs primarily perform two types of reactions: either (i) reducing the intermediate 3-\nketo-DON at the C3 ketone group to form 3-epi-DON, or (ii) oxidizing the C3-OH of\nDON to generate 3-keto-DON (AKR18A1, ref. 32). These are typical redox functions\nof AKRs (ref. 33), consistent with the canonical activity of this enzyme family.\nIn contrast, DONepi directly catalyzes the C3-epimerization of DON to form 3-epi-DON.\nThis epimerization activity is entirely distinct from the redox reactions of all previously\nreported DON-related AKRs—and to our knowledge, it represents the first\nidentification of an AKR with direct epimerization activity toward DON. More\nimportantly, we further resolved the underlying molecular mechanism of this\nepimerization (via cryo-EM and QM/MM simulations), which provides new insights\ninto AKR functional diversification beyond canonical redox roles.\nRegarding the multi-enzyme cascade study you referenced, its design and mechanism\nalso differ substantially from our work. That study employed three previously identified\nenzymes (two from our prior report: QDDH and AKR13B2 for stepwise C3-\nepimerization; one from their group: FHB7 for de-epoxidation) to achieve DON\ndegradation via a three-step cascade. In contrast, our work identifies a novel single-\nenzyme (DONepi) for direct C3-epimerization and pairs it with another newly\ndiscovered enzyme (DONrd for C8-reduction) to trigger a distinct pathway for absolute\nDON degradation. The core novelty here lies in the identification of two new enzymes\nwith non-canonical activities (epimerization for DONepi, C8-reduction for DONrd) and\nthe elucidation of their synergistic mechanism.\nWe acknowledge that the original manuscript may not have sufficiently highlighted\nthese critical distinctions, leading to potential misperception of novelty. In the revised\nmanuscript, we have strengthened the discussion section to explicitly contrast\nDONepi/DONrd with reported enzymes and pathways, with a focus on their unique\ncatalytic activities and mechanistic insights (Lines 329–332; Lines 342-343).\nWe believe these clarifications demonstrate that our study presents a novel DON\ndegradation mechanism, as you noted in the initial summary of our work, and hope this\naddresses your concern regarding novelty.\n2. Biosafety of the strain, it is essential to assess the biosafety of Nocardioides sp. S5-\n5 in industrial applications. Has the strain been screened for potential production of\nother harmful metabolites or toxins? Is there any genomic or metabolomic evidence\nsupporting its safety for animal or environmental use?\nResponse: We sincerely appreciate the reviewer's concern regarding the biosafety\nof Nocardioides sp. S5-5. Indeed, current knowledge on the biosafety aspects of this\nstrain—including potential production of harmful metabolites or toxins—is limited, and\ncomprehensive genomic or metabolomic data supporting its safety for animal or\nenvironmental applications remain insufficient.\nHowever, it’s crucial to note that the primary focus of our study is the discovery of\nnovel enzymes (DONepi and DONrd) derived from this strain. In future industrial\napplications, direct utilization of Nocardioides sp. S5-5 is not the intended approach.\nInstead, expression of these DON-degrading enzymes in well-characterized industrial\nchassis strains (e.g., E. coli, yeast) represents the planned and promising strategy for\ntheir practical implementation. These chassis organisms have established safety profiles\nand extensive use in industrial biotechnology. Therefore, while the safety\nof Nocardioides sp. S5-5 merits independent consideration, it is not directly relevant to\nthe future industrial implementation of the enzymes we identified—the core innovation\nof our work.\n3. The authors emphasize the practical potential of these enzymes; however, these\nenzymes still require co-factor NADP or NADPH to initiate the degradation. Therefore,\nthe authors did not adequately address the concerns they raised in the introduction\n(Lines 43-47), which remain unresolved in the current work.\nResponse：We appreciate the reviewer’s attention to the co-factor requirement of our\nidentified enzymes. As highlighted in Lines 44-48, a key limitation of previously\nreported DON epimerization detoxification systems is their dependence on the co-\nfactor pyrroloquinoline quinone (PQQ), which is necessary for the PQQ-dependent\nalcohol dehydrogenases that drive the first step of DON epimerization. PQQ is not only\nexpensive but also cannot be naturally synthesized by industrially relevant strains (e.g.,\nyeast, E. coli), severely restricting practical applications.\nIn contrast, the enzymes identified in our study (DONepi and DONrd) rely solely on\nNADP⁺ or NADPH as co-factors. Critically, these co-factors are naturally present and\nmetabolically accessible in the aforementioned industrial chassis strains and other\nbiological systems, eliminating the need for exogenous supplementation of costly co-\nfactors like PQQ. Thus, our work provides a more feasible path toward the industrial\nimplementation of DON-degrading enzymes, with clear relevance to resolving the co-\nfactor-related bottleneck outlined in the Introduction.\nWe hope these clarifications help address the concern regarding co-factor dependency\nand provide sufficient clarity on how our work contributes to addressing the previously\noutlined limitations.\n4. While the authors show that transgenic A. thaliana expressing DONepi and DONrd\nexhibit resistance to DON, this model system may lack translational relevance due to\nthe following reasons: 1) FHB primarily affects wheat, using transgenic wheat would\nprovide more substantial and more relevant evidence for agricultural applications; 2)\nDON toxicity is a significant concern in animals and humans via contaminated feed.\nTherefore, it would be more meaningful to evaluate the detoxification efficiency of\nthese enzymes or the strain in animal models or to simulate feed detoxification\nconditions.\nResponse：We sincerely appreciate the reviewer’s valuable comments.\n(1) Regarding the use of transgenic Arabidopsis thaliana for DON resistance analysis:\nDON detoxification genes are well-recognized as critical genetic resources for\nenhancing Fusarium head blight (FHB) resistance, as they can confer both\nresistance to pathogenic Fusarium spread and reduction in mycotoxin accumulation\nin crops (Mandala et al., MPMI, 2019, 32: 583-592; Wang et al., Nat. Commun.,\n2021, 12: 6426; He et al., Plant Biotech. J., 2024, 22: 2395-2409). While we\nacknowledge that FHB primarily affects wheat, using transgenic Arabidopsis to\nanalyze toxin tolerance is a widely adopted research strategy in the field. This\napproach helps evaluate the potential of candidate genes for FHB resistance\nimprovement (Poppenberger et al., J. Biol. Chem., 2003, 278:47905-47914; Shin et\nal., J. Exp. Bot., 2012, 63: 695-709; Wang et al., Nat. Commun., 2021, 12: 6426).\nImportantly, existing studies have demonstrated a high consistency between toxin\ntolerance phenotypes in Arabidopsis and FHB resistance phenotypes in crops (Shin\net al., J. Exp. Bot., 2012, 63: 695-709; Mandala et al., MPMI, 2019, 32: 583-592;\nWang et al., Nat. Commun., 2021, 12: 6426). Given that the core focus of this study\nis the identification and mechanistic characterization of novel detoxifying enzymes\n(DONepi and DONrd), our demonstration of DON tolerance in\ntransgenic Arabidopsis was intended to provide theoretical support for the future\napplication of these genes. We fully agree that your suggestion is highly\nconstructive. We are already conducting follow-up work to improve FHB resistance\nin cereal crops (e.g., wheat) using these detoxification genes, and this will be the\nfocus of our subsequent studies.\n(2) Regarding the evaluation of detoxification efficiency in animal models or under\nsimulated feed detoxification conditions: We greatly appreciate this insightful\nsuggestion. We recognize that verifying the efficacy of detoxifying enzymes in\nanimal models is essential before their large-scale application. For instance, in\nChina, authoritative regulatory bodies require long-term and systematic efficacy\nevaluations of enzymes in animal models (e.g., pigs and chickens) when applying\nfor new feed additive certifications. This work demands substantial time and\nsystematic design. Currently, we are actively advancing the relevant application\nprocedures; however, we believe this comprehensive animal-based evaluation\nexceeds the scope of the current study, which centers on enzyme discovery and\nmechanistic elucidation.\nWe hope this clarification conveys our commitment to addressing translational\nrelevance through follow-up work, and we thank you again for these valuable\nsuggestions that will guide our future research directions.\n5. The study focuses on two enzymes involved in the initial steps of DON degradation,\nbut the complete pathway of “absolute degradation” is not fully established. Does\nNocardioides sp further metabolize the products of DONepi and DONrd. S5-5?\nResponse (Lines 121–124; Lines 448-469; Supplementary Figures 11): Thank you\nfor your valuable comments. Nocardioides sp. S5-5 is indeed capable of further\nmetabolizing the products catalyzed by DONepi and DONrd. Evidence supporting this\nhas already been presented in the original manuscript: (1) Time-course analysis of DON\ndegradation by strain S5-5 (Figures 1f, 1g, and Supplementary Figure 2)\ndemonstrates the emergence and subsequent disappearance of the intermediates 3-epi-\nDON and 8-OH-DON. This confirms that these products are not accumulated but\nfurther metabolized. (2) Additional data (Supplementary Figures 3 and 37) show that\nstrain S5-5 can directly degrade 3-epi-DON and 8-OH-DON when these intermediates\nare used as sole substrates, respectively.\nFurthermore, to provide more direct evidence for the \"absolute degradation\" of DON,\nwe performed two new experiments, which measured CO production and the δ13C\n2\nvalues of respired CO when DON or stable carbon isotope (13C)-labelled DON was\n2\nused as the sole carbon source in MM medium. The results showed that: (1) In MM\nmedium with DON as the sole carbon source, a significant increase in CO levels was\n2\ndetected after strain S5-5 completely degraded DON (Supplementary Fig. 11a-c). (2)\nWhen 13C-labelled DON was used as the sole carbon source, substantial 13CO\n2\nproduction was observed following degradation by strain S5-5 (Supplementary Fig.\n11d, e).\nThese results collectively confirm that DON is indeed completely degraded by strain\nS5-5 into CO . We have added a brief description of this conclusion in the revised\nMaterials and Methods section (Lines 448-469). We hope these explanations and new\nexperimental data effectively address the reviewer's concerns.\nSupplementary Fig. 11. Detection of CO production following absolute\n2\ndegradation of DON by strain S5-5. DON and 13C-labelled DON were used as the sole\ncarbon sources in MM medium. At 7 days post inoculation (dpi) with strain S5-5, the concentration\nof CO and the δ13C content of respired CO were detected by gas chromatography (GC) and isotope\n2 2\nratio mass spectrometry (IRMS), respectively. a, HPLC analysis of DON levels in the medium at 7\ndpi. b, GC analysis of CO production at 7 dpi. c, Average CO concentration in samples at 7 dpi.\n2 2\nd, Representative ionic current intensities of masses 46, 45, and 44 (upper, middle, and bottom\npanels, respectively) acquired from IRMS for each treatment. e, δ13C values of respired CO in\n2\nsamples at 7 dpi. Data are from three independent biological experiments.\n6. Although the authors utilize cryo-EM and QM/MM simulations to study the catalytic\nmechanisms, the absence of substrate- or product-bound structures significantly limits\nmechanistic insights. Given that both enzymes are soluble and expressed in E. coli,\ndid the authors attempt to obtain X-ray structures with ligands? X-ray crystallography\noffers greater flexibility for soaking experiments and higher resolution, potentially\nfacilitating the visualization of key interactions with substrates or products.\nResponse (Lines 196-198): We sincerely appreciate your professional comments. To\nobtain substrate-bound protein structures, we have made multiple attempts, including\nX-ray crystallography; unfortunately, the full-length DONepi protein cannot be\ncrystallized either alone or with the DON ligand. This challenge appears to be common\nfor DON degrading enzymes, as previous structural studies also failed to obtain DON-\nbound crystals despite extensive efforts to do so via X-ray crystallography (Hu et al.,\nInt. J. Biol. Macromol., 2022, 200: 388-396; Yang et al., J. Agric. Food Chem., 2022,\n70: 6764-6774; Yang et al., JACS Au, 2024, 4: 619-634).\nWe therefore chose cryo-EM for structural analysis, as the enzyme naturally exists as a\nmultimer (~300 kDa), making it more suitable for this approach. Cryo-EM was widely\nused for structural analysis of various multimeric enzymes (Praharaj et al., Sci. Adv.,\n2025, 11: eads2664. Wenger and Christianson, Nat. Commun., 2025, 16: 5089).\nNevertheless, we didn’t capture the substrate-bound state with cryo-EM, likely due to\nthe weak binding affinity between the protein and substrate. To overcome this limitation,\nwe have clarified the key active sites of DONepi, including Y27, R86 and the catalytic\ntetrad (D51, Y56, K83, H124), and further investigated its catalytic mechanism by using\nmultiple simulation approaches and site-directed mutagenesis. By integrating cryo-EM,\nQM/MM simulations, and site-directed mutagenesis, we provided complementary\ninsights that together elucidate the catalytic mechanisms of DONepi: The octameric-\nstructured DONepi alone catalyzes C3-epimerization of DON by steering the rigid-\nbody rotation of the transient 3-keto intermediate for stereoinverting reduction. To our\nknowledge, these methods are among the most well-established in current\nenzymological mechanism research. (Brog et al., Angew. Chem. Int. Ed., 2023, 62:\ne202211937; Wang et al., Science, 2024, 384: 1453-1460).\nTo address this concern more explicitly, we have added the following statement to the\nResults section of the revised manuscript (Lines 196-198): “Regrettably, despite\nmultiple attempts, we have not been able to obtain protein structure data with bound\nsubstrates, which may be attributed to weak binding affinity between the protein and\nsubstrate, among other potential factors.”\nWe hope this clarification effectively addresses your concern.\nOther minor concerns and suggestions for the authors to consider:\n1. The introduction provides a summary of DON-degrading enzymes, but several\nimportant and relevant studies are missing. The authors should consider citing:\nenzymatic degradation of T-2 toxin and DON (Yang J, https://doi.org/10.1021\n/acscatal. 5c03462; Huang J, https://www.nature.com/articles/s41589-019-0446-8); An\ninteresting and similar strategy to target epoxy group and 3-keto using a multi-enzyme\ncascade reaction (Hou B, https://doi.org/10.1016/j. foodchem.2024.139886);\nResponse (Lines 30-34; refs. 12, 14, 19-21): We sincerely appreciate your valuable\nsuggestion to supplement relevant citations, which helps improve the\ncomprehensiveness of our introduction. We have now cited these studies in\nthe appropriate place in the revised manuscript. The three studies you mentioned are\nimportant and interesting. We have been paying attention to this research area, and we\nwould like to clarify the context of their citation in our manuscript:\n(1) For the study by Yang et al. (ACS Catal., 2025, 15: 11664-11672): This work\nfocuses on developing enzymes derived from Fhb7 that are capable of T-2\ndegradation via ancestral sequence reconstruction (ref. 14). Actually, we have\nalready cited the original foundational studies on the DON degradation activity of\nFHB7 in our manuscript (refs. 6 and 13). Additionally, we note that this derivative\nenzyme study was published on June 20, 2025—after the submission of our current\nmanuscript—making it unavailable for inclusion during our initial literature review.\n(2) For the study by Huang et al. (Nat. Chem. Biol., 2020, 16: 250-256): We have been\npaying attention to this research since its publication. Actually, the authors stated\nthat the protein studied in this paper, specialized glyoxalase (SPG), catalyzes the\ntransformation of 3-acetyl-deoxynivalenol (3-Ac-DON) but not DON. Given its\nlack of direct relevance to the “DON-degrading enzymes” theme of our introduction,\nwe did not initially cite it. However, recent studies (especially one published just\nlast month) have demonstrated that SPG does exhibit DON transformation activity\n(Hu et al., Int. J. Biol. Macromol., 2022, 200: 388-396; Lee et al., Adv. Sci., 2025,\n12: e02914), and these recent findings have also been cited in the revised\nmanuscript (refs. 19-21).\n(3) For the study by Hou et al. (Food Chem., 2024; 456: 139886): This work reports a\nthree-step catalytic reaction for DON degradation using three previously identified\nDON-degrading enzymes (QDDH, AKR13B2, and FHB7). We had already cited\nthe original studies that first characterized these three enzymes in the original\nmanuscript (refs. 6 and 24) to acknowledge their foundational roles. Now, we\nhave included it in the revised manuscript to further strengthen the context of our\nintroduction (ref. 12).\nWe hope this clarification addresses your concern about literature coverage.\n2. The manuscript lacks kinetic parameters for DONepi and DONrd. Determining\nparameters such as kcat, Km, and catalytic efficiency would significantly strengthen\nthe characterization of these enzymes.\nResponse (Lines 138-143): Thank you very much for your valuable suggestions, which\nhave been incredibly insightful for us to refine our research work. We have followed\nyour advice and conducted additional experiments to determine the kinetic parameters\nof DONepi and DONrd, including k , K , and catalytic efficiency (k /K ). These\ncat m cat m\ncrucial kinetic data have been integrated into the revised manuscript (see Lines 138-\n143 in the Results section). We believe that these supplementary data not only address\nyour concerns but also significantly enhance the characterization of these enzymes.\nOnce again, thank you for your constructive feedback.\n3. Line 95, “24 hai” appears to be a typographical error. Should this be “24 h”?\nResponse (Line 97): The abbreviation “hai” here stands for \"hours after inoculation,\"\nwhich was used to specify the time point post-inoculation. To avoid ambiguity, we have\nadded a clear explanation of this abbreviation (hours after inoculation) in the revised\nmanuscript. We appreciate your attention to this detail, which has helped us improve\nthe readability of our text.\n4. In Lines 36–37, the authors state that 3-epi-DON has the lowest toxicity among DON\nmetabolites based on a 2015 reference. However, recent studies targeting the epoxy\ngroup (a major contributor to DON toxicity) have shown promising detoxification\noutcomes. The authors should discuss whether 3-epi-DON remains less toxic than\nmetabolites derived from the degradation of the epoxy group.\nResponse (Lines 37-38): Thank you for your insightful comments. It is well-\nestablished that the C3-OH and C12,13-epoxy groups of DON are the primary\ncontributors to its toxicity. 3-epi-DON and dE-DON are two well-studied microbial\ndetoxification metabolites, formed by modifications of the C3-OH and C12,13-epoxy\ngroups, respectively.\nAccording to published data, the IC values of 3-epi-DON (281 μg/mL) and dE-DON\n50\n(23.3 μg/mL) are 1181-fold and 54-fold higher than that of DON (0.23 μg/mL and 0.44\nμg/mL, respectively) in the BrdU bioassay (Eriksen et al., Food Chem. Toxicol., 2004,\n42: 619-624; He et al., Food Chem. Toxicol., 2015, 84: 250-259). These findings are\nwidely cited in the field (with 224 and 104 citations, respectively). While dE-DON is\noften described as a low-toxicity DON metabolite, several studies have shown it\nexhibits greater biological activity than previously hypothesized, including negative\neffects on cattle reproduction and immune-modulatory effects similar to DON in piglets\n(Guerrero-Netro et al., Biol. Reprod., 2017, 97: 746-757; Pierron et al., Arch. Toxicol.,\n2018, 92: 3381-3389; Guerrero-Netro et al., J. Appl. Toxicol., 2020, 41: 1180-1187). In\ncontrast, adverse effects of 3-epi-DON have rarely been reported to date.\nMore recently, Hou et al. (Food Chem., 2024, 456: 139886) reported different BrdU\nbioassay results: the IC values of 3-epi-DON (77.5 μg/mL) and dE-DON (127 μg/mL)\n50\nare 259-fold and 449-fold higher than that of DON (0.30 μg/mL), respectively.\nAdditionally, DON-13-GSH, which is another metabolite formed by glutathionylation\nof DON’s C12,13-epoxy group via Fhb7-GST (a glutathione S-transferase\nfrom Thinopyrum ponticum), also exhibits high IC values (962-fold higher than DON)\n50\n(Hou et al., Food Chem., 2024, 456: 139886). We recognize this discrepancy in\npublished data likely underpins the reviewer’s concern.\nGiven the inconsistent data on toxicity comparisons between 3-epi-DON and\nmetabolites derived from C12,13-epoxy group modification, it is difficult to\ndefinitively conclude which is less toxic. For rigor, we have revised the sentence in\nLines 37-38 to: “The final product of DON C3-epimerization, 3-epi-DON, is one of\nthe lowest-toxicity metabolites among the currently known microbial metabolites of\nDON”. Due to the focus and length constraints of this article, we have not expanded on\nthis topic in the main text, but we hope this revised, cautious statement addresses your\nconcern.\n5. In Supplementary Table 4 and Methods (Lines 466–469), the authors do not provide\ninformation on the initial model used for DONepi structure determination. Clarifying\nthe template and method used for initial model building (e.g., AlphaFold prediction,\nhomology modeling) is essential for reproducibility and transparency.\nResponse (Line 539; Supplementary Table 4): Thank you for your insightful\ncomments. As per your suggestion, we have added a brief description in the Methods\nsection to clarify the template and method used for initial model building (Line 539).\nWe have also updated the relevant information in Supplementary Table 4."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors are to be commended for addressing the majority of the reviewers' concerns\nMajor issues\n1. While the revised manuscript shows substantial improvement, it still lacks evidence supporting the hypothesis that DONepi catalyzes a two-step reaction via an enzyme-bound 3-ketoDON intermediate. The time-course analyses for DON epimerization and NADPH formation were conducted over several hours; however, detecting transient intermediates would be more feasible if the assays were performed on the timescale of seconds or minutes. For example, could the NADPH assay be adapted into a continuous spectrophotometric format to monitor changes in real time over a shorter duration?\n2. The new data presented in the revised manuscript also raise the possibility of alternative mechanisms that could explain the absence of detectable 3-ketoDON in the assay. The overall activity of DONepi is very low (kcat = 0.3 min⁻¹). Could it be that the initial transformation of DON to 3-ketoDON is significantly slower than the subsequent conversion of 3-ketoDON to 3-epiDON? If so, any 3-ketoDON that is produced and released by the enzyme may be rapidly converted to 3-epiDON, hence the absence of detectable 3-ketoDON.\n3. The overall low turnover rate of DONepi also raises questions about its potential biotechnological utility for DON detoxification, given its limited catalytic efficiency.\nMinor issues\n1. Source of C13 labelled DON should be indicated. Also are all carbon atoms labelled ?\n2. Concentration of coenzyme used in enzymes assays should be provided", "reviewer_2": "I appreciate the authors' efforts in revising the manuscript and for providing substantial new data that has significantly strengthened the study. The work presents an interesting and valuable enzymatic strategy for DON degradation, and I believe it has the potential to be suitable for publication in Nature Communications once the following points are adequately addressed.\n1. Novelty Claim Regarding Epimerization Activity: The authors claim that DONepi represents \"the first identification of an AKR with direct epimerization activity toward DON.\" However, this claim appears to be inaccurate in light of a recent publication (DOI: 10.1016/j.fct.2024.115159), which also reports an AKR with direct epimerization activity on DON. Although the paper has not elucidated the catalytic mechanism, the fundamental activity has been previously described. The authors must temper their novelty statement, properly cite this prior work and strengthen the manuscript by including a comparative analysis, such as unique structural motifs, catalytic residues, or superior kinetic parameters, will be crucial to establishing the distinct contribution of this study.\n2. Interpretation of CO₂ Production Data: In the revised experiments measuring CO₂ production with DON or ¹³C-labelled DON as the sole carbon source, the high background level of CO₂ (exceeding 2500 ppm) in the no-carbon source control is concerning. This result suggests that strain S5-5 produces a significant amount of CO₂ independently of the supplied carbon source. The authors must clarify the origin of this background CO₂.\n3. Source and Characterization of ¹³C-Labelled DON: The manuscript should include a detailed description of the source and synthesis of the ¹³C-labelled DON used in this study. If it was commercially procured, the vendor and catalog number should be specified. If it was chemically synthesized, the authors should provide the synthetic route and full characterization data (e.g., NMR spectra, etc) in the Supplementary Information to verify the compound's identity and isotopic purity, which is critical for the conclusions drawn from the labelling experiments.\n4. Enzyme Kinetics Data Presentation: The raw data for the kinetic characterization (e.g., the plot of velocity vs. substrate concentration) should be included in the Supplementary Figures. Furthermore, essential experimental details for the kinetic assays, such as the concentration ranges of the substrates and co-factors used, must be explicitly stated in the Methods section or the corresponding figure legends.\n5. Line 308-309: The statement suggesting the involvement of a \"potential cytochrome P450\" in further DON degradation is speculative without supporting evidence. As many non-P450 oxidases are capable of catalyzing hydroxylation reactions, using the more general term \"oxidase\" would be more accurate and appropriate here.\n6. Please correct the typographical error on Line 449: \"OD600value\" should be revised to \"OD600 value\"."}, "author_response": "A list of responses to reviewers\nWe thank the anonymous referees for their time and thoughtful suggestions to help to\nfurther improve our manuscript and for identifying errors in the writing and Figure\npresentation. We have made all changes/responses based on the advice and\ncomments/suggestions made by the reviewers. All changes, as well as our responses,\nare outlined below as they appear in the order of the comments and suggestions.\nReviewer #1 (Remarks to the Author):\nThe authors are to be commended for addressing the majority of the reviewers' concerns\nMajor issues\n1. While the revised manuscript shows substantial improvement, it still lacks evidence\nsupporting the hypothesis that DONepi catalyzes a two-step reaction via an enzyme-\nbound 3-ketoDON intermediate. The time-course analyses for DON epimerization and\nNADPH formation were conducted over several hours; however, detecting transient\nintermediates would be more feasible if the assays were performed on the timescale of\nseconds or minutes. For example, could the NADPH assay be adapted into a continuous\nspectrophotometric format to monitor changes in real time over a shorter duration?\nResponse: Thank you for your careful guidance. To address your concern regarding the\ndetection of NADPH, we have adapted a continuous spectrophotometric assay to\nmonitor it in real time with a minute-scale time resolution, using a Spark 10M\nMultimode Plate Reader (Tecan, Männedorf, Switzerland). This monitoring was\nconducted with more than 1000 measurements taken at 1-minute intervals. Regrettably,\nas shown in the figures below, we did not detect significant NADPH formation.\nThis outcome is consistent with your speculation in the subsequent comment: the initial\ntransformation of DON to 3-keto DON is significantly slower than the subsequent\nconversion of 3-keto DON to 3-epi DON, meaning any NADPH produced during the\noxidation of DON to 3-keto DON by DONepi is rapidly converted to NADP+ during\nthe reduction of 3-keto DON to 3-epi DON. Furthermore, our previously provided\nexperimental results (Supplementary Fig. 33) confirm that NADPH serves as the\ncoenzyme for the reduction of 3-keto DON to 3-epi DON by DONepi. These reaction\nprocesses are also supported by QM/MM metadynamics simulations (Fig. 4;\nSupplementary Figs. 31 and 32; Supplementary Movies 1 and 2). Similarly,\nBcUGAepi-NADH is also below the detection limit during the C4-epimerization of\nUDP-glucuronic acid (UDP-GlcA) by BcUGAepi, which uses NAD+ as a coenzyme\nand employs a transient 4-keto intermediate that is not released from the enzyme to\nachieve epimerization (Borg et al., FEBS J. 2021, 288: 1163-1178; Borg et al., Angew.\nChem. Int. Ed. 2023, 62: e202211937).\nWe hope that the supplementary experiment conducted as suggested and the\ncomprehensive explanations based on experimental, simulation, and literature evidence\nwill address your concerns adequately.\nFigure A1. Continuous spectrophotometric analysis of NADPH formation during\nDON epimerization by DONepi with NADP⁺ as the coenzyme. Time-course detection\nof the optical density at 340 nm (characteristic of NADPH) was performed at 1-minute intervals\nafter incubation with DONepi. Values are presented as the means of five replicates; error bars\nrepresent standard deviation. a, The optical density at 340 nm during 2-9 hours after incubation\n(hai), from 480 consecutive measurements. b, The optical density at 340 nm during 12-19 hai, from\n480 consecutive measurements. c-f, The optical density at 340 nm at 1, 6, 12, 18 hai, from 60\nconsecutive measurements, respectively.\n2. The new data presented in the revised manuscript also raise the possibility of\nalternative mechanisms that could explain the absence of detectable 3-ketoDON in the\nassay. The overall activity of DONepi is very low (kcat = 0.3 min⁻¹). Could it be that\nthe initial transformation of DON to 3-ketoDON is significantly slower than the\nsubsequent conversion of 3-ketoDON to 3-epiDON? If so, any 3-ketoDON that is\nproduced and released by the enzyme may be rapidly converted to 3-epiDON, hence\nthe absence of detectable 3-ketoDON.\nResponse: We appreciate the reviewer’s insightful and professional perspective.\nSpecifically, according to our multiscale simulation results, the formation of the\ntransient 3-keto DON intermediate from DON has a free energy barrier of 11.8 kcal/mol\n(Fig. 4b), whereas the free energy barrier for the conversion of 3-keto DON to 3-epi\nDON is less than 2.5 kcal/mol (Fig. 4f). The conversion of the transient 3-keto DON\nintermediate to 3-epi DON is much easier than the formation of the 3-keto DON\nintermediate, hence the absence of detectable 3-keto DON. A similar phenomenon has\nalso been reported in previous studies on UDP-glucuronic acid 4-epimerase: the\nreduction of the keto intermediate is considerably faster than its formation, so no 4-keto\nintermediate accumulates, which is consistent with the observation of undetectable 3-\nketo DON (Borg et al., FEBS J. 2021, 288: 1163-1178).\n3. The overall low turnover rate of DONepi also raises questions about its potential\nbiotechnological utility for DON detoxification, given its limited catalytic efficiency.\nResponse: Thank you for your comment. We deeply understand your concern about\nthe potential biotechnological utility of DONepi for DON detoxification, given its\nlimited catalytic efficiency. Indeed, several enzymes with degradation activity on DON\nand its structural analogs reported in recent studies exhibit limited catalytic efficiency,\nsuch as the newly discovered extant SPG (specialized glyoxalase I) enzyme OR9\ntowards DON (k = 2.8 ± 0.7 min-1) and the FHB7 (glutathione-S transferase) variant\ncat\nM10 towards T-2 toxin (k = 2.0 ± 0.1 min-1) (Lee et al., Adv. Sci., 2025, 12: e02914;\ncat\nYang et al., ACS Catal., 2025, 15: 11664-11672). This reflects that enzymatic\ndegradation of DON and its structural analogs is inherently challenging. However,\nDONepi and all these reported enzymes represent new avenues for DON degradation\nand provide a foundation for further improving catalytic efficiency. Notably, the\nproduct of DON C3-epimerization by DONepi, 3-epi-DON, is one of the lowest-\ntoxicity metabolites among the currently known microbial metabolites of DON, with a\n1181-fold decrease in toxicity. Fortunately, engineering of mycotoxin-degrading\nenzymes via protein engineering methods (e.g., ancestral sequence reconstruction) has\nbeen successfully used to enhance catalytic efficiency (Lee et al., Adv. Sci., 2025, 12:\ne02914; Yang et al., ACS Catal., 2025, 15: 11664-11672). Moreover, the rapidly\ndeveloping AI-informed approach will facilitate easier improvement of catalytic\nefficiency for such enzymes through artificial protein evolution (Fei et al., Cell, 2025,\n188: 1-19). In future work, we will focus on optimizing the catalytic efficiency of\nDONepi to meet the requirements for its practical application in DON detoxification.\nWe hope these explanations, supporting references, and future research plans\nadequately address your concern.\nMinor issues\n1. Source of C13 labelled DON should be indicated. Also are all carbon atoms labelled ?\nResponse (Lines 405-406): Thank you for your careful guidance. The 13C-labelled\nDON used in this study was commercially procured from Alta Scientific Co., Ltd.\n(Tianjin, China), with the catalog number 1ST7213C15-25A. Regarding the isotopic\nlabelling information, all carbon atoms of the 13C-labelled DON are 13C-labelled, as\nspecified by the vendor. To clarify these details, we have added detailed information of\nthe 13C-labelled DON (including vendor and catalog number) in the Materials and\nMethods section (Lines 405-406).\n2. Concentration of coenzyme used in enzymes assays should be provided\nResponse (Lines 503-504, Supplementary Figs. 16b and 17b): Thank you for your\ncomment. The detailed concentration of the coenzyme used in the enzyme assays has\nbeen provided in the Materials and Methods section (Lines 503-504). In addition, it has\nalso been explicitly stated in the corresponding figure legends of the newly included\nSupplementary Figs. 16b and 17b, which represent the kinetic curves of the two\nenzymes with DON, respectively.\nReviewer #2 (Remarks to the Author):\nI appreciate the authors' efforts in revising the manuscript and for providing substantial\nnew data that has significantly strengthened the study. The work presents an interesting\nand valuable enzymatic strategy for DON degradation, and I believe it has the potential\nto be suitable for publication in Nature Communications once the following points are\nadequately addressed.\n1. Novelty Claim Regarding Epimerization Activity: The authors claim that DONepi\nrepresents \"the first identification of an AKR with direct epimerization activity toward\nDON.\" However, this claim appears to be inaccurate in light of a recent publication\n(DOI: 10.1016/j.fct.2024.115159), which also reports an AKR with direct\nepimerization activity on DON. Although the paper has not elucidated the catalytic\nmechanism, the fundamental activity has been previously described. The authors must\ntemper their novelty statement, properly cite this prior work and strengthen the\nmanuscript by including a comparative analysis, such as unique structural motifs,\ncatalytic residues, or superior kinetic parameters, will be crucial to establishing the\ndistinct contribution of this study.\nResponse (Lines 333-336; Supplementary Fig. 38): Thank you for your valuable\ncomments and guidance to improve the accuracy of our novelty statement and\nmanuscript rigor.\nAs per your suggestion, we performed a comparative analysis of amino acid sequences\nbetween DONepi and AKR11A2 (DOI: 10.1016/j.fct.2024.115159), the previously\nreported AKR with DON epimerization activity. The results show low amino acid\nsequence identity (less than 32%) between the two enzymes, implying a distinct\nevolutionary origin. While both proteins contain the conserved D-Y-K-H catalytic\ntetrad typical of the AKR superfamily, further detailed comparison (e.g., kinetic\nproperties, DON-binding modes, critical catalytic residues) is not feasible due to the\nlack of such data in the prior study. In contrast, our work comprehensively demonstrates\nthe uniqueness of DONepi through multi-dimensional analyses, including phylogenetic\nclassification, cryo-EM structural characterization, and elucidation of the catalytic\nmechanism. These findings highlight the distinct contribution of our study beyond the\nprior report.\nTo address this comparison explicitly, we have added Supplementary Fig. 38 (amino\nacid sequence alignment and similarity analysis of DONepi and AKR11A2) and a\ncorresponding description in the Discussion section (Lines 333-336) to clarify the\ndifferences between the two enzymes. In addition, a new reference has been cited as\nRef. 33.\n2. Interpretation of CO₂ Production Data: In the revised experiments measuring CO₂\nproduction with DON or ¹³C-labelled DON as the sole carbon source, the high\nbackground level of CO₂ (exceeding 2500 ppm) in the no-carbon source control is\nconcerning. This result suggests that strain S5-5 produces a significant amount of CO₂\nindependently of the supplied carbon source. The authors must clarify the origin of this\nbackground CO₂.\nResponse: Thank you for your valuable comments. Indeed, a high background level of\nCO was detected in the control group without additional DON (only strain S5-5 in\n2\nminimal medium, MM). We believe this background CO should originate from\n2\nresidual carbon sources in the nutrient broth (NB) medium used for pre-culturing strain\nS5-5. As described in the Materials and Methods section, strain S5-5 was first cultured\nin NB medium before being transferred into MM. Although the bacterial cells were\nwashed three times with sterilized water prior to resuspension in MM, some\nunassimilated carbon sources may still remain in the cells, which likely contributes to\nthe high CO background in the strain-only control.\n2\nwith DON (strain S5-5+DON in MM). Furthermore, to eliminate the interference of\nbackground CO , we used 13C-labelled DON as the sole carbon source in MM, and\n2\nobvious production of 13CO was detected. These results collectively demonstrate that\n3. Source and Characterization of ¹³C-Labelled DON: The manuscript should include a\ndetailed description of the source and synthesis of the ¹³C-labelled DON used in this\nstudy. If it was commercially procured, the vendor and catalog number should be\nspecified. If it was chemically synthesized, the authors should provide the synthetic\nroute and full characterization data (e.g., NMR spectra, etc) in the Supplementary\nInformation to verify the compound's identity and isotopic purity, which is critical for\nthe conclusions drawn from the labelling experiments.\nResponse (Lines 405-406): Thank you for your valuable guidance. The 13C-labelled\nDON used in this study was commercially procured from Alta Scientific Co., Ltd.\n(Tianjin, China), with the catalog number 1ST7213C15-25A. As per your suggestion,\nwe have added detailed information of the 13C-labelled DON (including vendor and\ncatalog number) in the Materials and Methods section (Lines 405-406).\n4. Enzyme Kinetics Data Presentation: The raw data for the kinetic characterization\n(e.g., the plot of velocity vs. substrate concentration) should be included in the\nSupplementary Figures. Furthermore, essential experimental details for the kinetic\nassays, such as the concentration ranges of the substrates and co-factors used, must be\nexplicitly stated in the Methods section or the corresponding figure legends.\nResponse (Supplementary Figs. 16b, 17b): Thank you for your insightful comments\nto improve the completeness of our kinetic data presentation. We have included the raw\ndata for the kinetic characterization of two enzymes as Supplementary Figs. 16b and\n17b. In addition, essential experimental details for the kinetic assays, including the\nconcentration ranges of substrates and co-factors, have been explicitly stated in the\ncorresponding figure legends of Supplementary Fig. 16b and 17b.\n5. Line 308-309: The statement suggesting the involvement of a \"potential cytochrome\nP450\" in further DON degradation is speculative without supporting evidence. As many\nnon-P450 oxidases are capable of catalyzing hydroxylation reactions, using the more\ngeneral term \"oxidase\" would be more accurate and appropriate here.\nResponse (Lines 14, 309-310, 357-358, 897; Fig. 6): Thank you for pointing out this\ndetail and guiding us to refine the accuracy of our statement. We highly appreciate your\nrigorous approach to scientific expression. As suggested, we have revised the\nspeculative term “cytochrome P450” to the more general and accurate “a potential\noxidase (e.g., cytochrome P450 enzyme)” in the revised manuscript (Lines 14, 309-310,\n357-358, 897; Fig. 6).\n6. Please correct the typographical error on Line 449: \"OD600value\" should be revised\nto \"OD600 value\".\nResponse (Line 455): Thank you for your careful check and attention to detail. We\napologize for the carelessness that led to this typographical error. As noted, we have\nrevised \"OD600value\" to \"OD value\" in Line 455 of the manuscript.\n600"}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have not adequately addressed the major concerns raised by reviewers of the previous manuscript.\nThis study describes DONepi, an enzyme that converts the mycotoxin DON into the less toxic metabolite 3-epiDON through a two-step reaction. DON is first oxidized to 3-ketoDON, which is then reduced to 3-epiDON. Based on molecular dynamics simulations, the authors propose that the 3-ketoDON intermediate remains bound within the enzyme’s active site, changes orientation, and is subsequently converted to 3-epiDON. The primary experimental observation in support of this mechanism is the absence of detectable 3-ketoDON during the reaction.\nHowever, new experimental data presented by the authors provides an alternative explanation. The initial conversion of DON to 3-ketoDON is considerably slower than the subsequent reduction of 3-ketoDON to 3-epiDON. Therefore, the lack of detectable 3-ketoDON may simply reflect that only a small amount of this intermediate is formed at any one time and is rapidly consumed in the second reaction. It remains possible that 3-ketoDON is released from the active site and subsequently rebinds the enzyme for subsequent conversion to 3-epiDON. Without additional experimental validation, the authors cannot conclusively support the proposed mechanism of 3-keotDON intermediate retention in the catalytic mechanism.\nA second issue concerns the catalytic performance of DONepi itself. The enzyme exhibits a low turnover rate (kcat = 0.3 min⁻¹), which substantially limits its practical biotechnological utility. Other reported enzymes capable of catalyzing the same reaction display significantly higher turnover rates, such as DepA (1.69 s⁻¹; Toxins 2024 Mar 5;16(3):136) and DepB 3 s-1 (Sci Rep. 2022 Aug 30;12:14737). This comparison highlights that the enzyme, in its current form, is markedly less efficient than existing alternatives.\nGiven these unresolved issues, I am unable to recommend this manuscript in the present form for publication.", "reviewer_2": "All of my previous concerns have been addressed."}, "author_response": "A list of responses to the reviewer\nWe thank the anonymous referee very much for helping to improve our manuscript. We\nhave implemented all revisions and provided corresponding responses in light of the\nreviewer’s advice and comments. All revisions and responses are listed below in the\norder of the reviewer’s comments.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have not adequately addressed the major concerns raised by reviewers of\nthe previous manuscript.\n(1) This study describes DONepi, an enzyme that converts the mycotoxin DON into the\nless toxic metabolite 3-epiDON through a two-step reaction. DON is first oxidized to\n3-ketoDON, which is then reduced to 3-epiDON. Based on molecular dynamics\nsimulations, the authors propose that the 3-ketoDON intermediate remains bound\nwithin the enzyme’s active site, changes orientation, and is subsequently converted to\n3-epiDON. The primary experimental observation in support of this mechanism is the\nabsence of detectable 3-ketoDON during the reaction.\nHowever, new experimental data presented by the authors provides an alternative\nexplanation. The initial conversion of DON to 3-ketoDON is considerably slower than\nthe subsequent reduction of 3-ketoDON to 3-epiDON. Therefore, the lack of detectable\n3-ketoDON may simply reflect that only a small amount of this intermediate is formed\nat any one time and is rapidly consumed in the second reaction. It remains possible that\n3-ketoDON is released from the active site and subsequently rebinds the enzyme for\nsubsequent conversion to 3-epiDON. Without additional experimental validation, the\nauthors cannot conclusively support the proposed mechanism of 3-keotDON\nintermediate retention in the catalytic mechanism.\nResponse (Lines 252-261; Lines 373-386; Supplementary Figs. 34 and 40): Thank\nyou for the careful guidance on improving our study and manuscript. To validate the\nproposed mechanism of 3-keto-DON intermediate retention in DONepi-catalyzed\nreactions, we have performed competitive nucleophilic assays using hydroxylamine as\na strong nucleophile, and the results confirm that the 3-keto-DON intermediate is not\nreleased from the enzyme active site. In addition, we have conducted structural and\nevolutionary conservation analyses, which further verify the structural basis underlying\nthis intermediate-retention mechanism of DONepi.\nFirst, the reduction of the intermediate 3-keto-DON catalyzed by DONepi depends on\nthe nucleophilic attack of the hydride (H-) from NADPH on the electrophilic C3\ncarbonyl carbon of 3-keto-DON. To test whether 3-keto-DON is released from the\nenzyme active site during catalysis, we used hydroxylamine hydrochloride\n(NH OH·HCl)—a reagent with stronger nucleophilicity than NADPH—as a\n2\ncompetitive probe to target the carbonyl group of 3-keto-DON. When hydroxylamine\n(1 mM) was added to the DONepi-catalyzed reaction system using 3-keto-DON as the\nsubstrate (50 mM sodium phosphate buffer, pH 7.0, 37°C), it reacted competitively with\n3-keto-DON. As confirmed by HPLC analysis, the product was almost exclusively the\noxime adduct of 3-keto-DON and hydroxylamine (Supplementary Fig. 34a, c).\nThis result confirms that hydroxylamine can readily outcompete NADPH in this reaction,\nleading to almost complete oximation of free 3-keto-DON without undergoing\nenzymatic epimerization. In contrast, when hydroxylamine (1 mM) was added to the\nDONepi-catalyzed reaction system using DON as the substrate (under identical buffer\nand temperature conditions), the product detected was still 3-epi-DON, which is\nconsistent with the reaction system without hydroxylamine (Supplementary Fig. 34b).\nThese data validate that the intermediate 3-keto-DON is not released from the active\nsite during the DON epimerization process catalyzed by DONepi. Instead, 3-keto-DON\nremains bound within the enzyme active site, where it is specifically reduced by\nNADPH to form 3-epi-DON without accessing external nucleophilic reagents.\nSecond, to further corroborate this conclusion from a structural perspective, we\nanalyzed the structural and evolutionary conservation of DONepi with SDR-type\nepimerases, which are well-characterized enzymes that employ a conserved “transient\nketo intermediate” mechanism (Kavanagh et al., Cell. Mol. Life Sci., 2008, 65: 3895-\n3906; Van Overtveldt et al., Biotechnol. Adv., 2015, 33:1814-1828; Fushinobu, Curr.\nOpin. Chem. Biol., 2021, 61: 53-62). Our results revealed that despite the lack of\nsignificant amino acid sequence similarity (Supplementary Fig. 40a), DONepi\nexhibits convergent evolution in active site conformation with SDR-type epimerases.\nSpecifically, the catalytically critical residues, which are Asp-Tyr-Lys-His in DONepi\nand Ser/Thr-Tyr-Lys in SDR-type epimerases, can be nearly superimposed in their 3D\nstructures (Supplementary Fig. 40b). This structural convergence strongly suggests\nthat DONepi shares a similar epimerization mechanism with SDR-type epimerases.\nNotably, the conserved “transient keto intermediate” mechanism in these well-studied\nSDR-type epimerases is characterized by the following key features: the reaction\ninitiates with hydride transfer to NAD(P)+, forming a transient keto intermediate.\nThis intermediate then undergoes rotation within the active site, followed by hydride back-\ntransfer from NAD(P)H to the carbonyl carbon, thereby completing the epimerization.\nThe reduction of the generated keto intermediate is considerably faster than its\nformation, which effectively prevents its accumulation or release from the enzyme\nactive site (Iacovino et al., J. Biol. Chem., 2020, 295: 12461-12473; Borg et al., FEBS\nJ., 2021, 288: 1163-1178; Borg et al., Angew. Chem. Int. Ed., 2023, 62: e202211937).\nOur experimental and computational data show strong similarity to this conserved\nmechanism.\nWe now have included two paragraphs of detailed description about these supplemented\nexperimental and analytical results in the revised Results and Discussion sections to\naddress this critical mechanistic concern explicitly (Lines 252-262; Lines 373-383). In\naddition, five new references supporting the transient keto intermediate mechanism\nhave been cited as Refs. 36-40.\nCollectively, the newly supplemented competitive nucleophilic assays and\nstructural/evolutionary conservation analyses, together with the additional experiments\nof 3-keto-DON and NADPH detection presented in our two previous rounds of revision,\nand the integrated evidence from in vitro enzyme assays, cryo-EM structural\ndetermination and molecular dynamics simulations reported in the manuscript, provide\ncompelling evidence that DONepi employs the transient keto intermediate mechanism,\nin which the 3-keto-DON intermediate remains enzyme-bound and is rapidly converted\nto 3-epi-DON without being released from the enzyme active site.\nSupplementary Fig. 34. Competitive nucleophilic reaction of hydroxylamine with\n3-keto-DON. Hydroxylamine (1 mM) was added to the DONepi-catalyzed reaction system using\neither 3-keto-DON or DON as the substrate (50 mM sodium phosphate buffer, pH 7.0, 37 °C). Each\nexperiment included four treatment groups: (1) DONepi-only group: reaction system containing\nDONepi but no hydroxylamine; (2) DONepi + hydroxylamine group: reaction system with both\nDONepi and hydroxylamine; (3) hydroxylamine-only group: reaction system containing\nhydroxylamine but no DONepi; (4) control group: reaction system with neither DONepi nor\nhydroxylamine. a, Hydroxylamine supplementation in the DONepi-catalyzed reaction system with\n3-keto-DON as the substrate. 3-keto-DON, its enzymatic product 3-epi-DON, and its\nhydroxylamine adduct 3-keto-DON oxime were detected via HPLC. b, Hydroxylamine\nsupplementation in the DONepi-catalyzed reaction system with DON as the substrate. DON and its\nenzymatic product 3-epi-DON were detected via HPLC. c, GC-MS analysis of 3-keto-DON oxime.\nThe compound was derivatized using a mixture of trimethylsilyl imidazole/trimethylchlorosilane\n(100:1, v/v). Extracted ion chromatograms (left panel) and corresponding mass spectra (right panel)\nof the trimethylsilyl (TMSI)-derivatized product are presented. Insets show the molecular structure\nof 3-keto-DON oxime (molecular weight, MW = 309.3 Da).\nSupplementary Fig. 40. Analysis of structural and evolutionary conservation of\nDONepi with SDR-type epimerases. a, Multiple sequence alignment of DONepi with\nrepresentative SDR-type epimerases whose protein crystal structures have been elucidated. The\nletters “e” and “h” at the bottom of the diagram respectively denote the conserved secondary\nstructures of the β-strand and the α-helix. The conserved catalytic tetrad (Asp-Tyr-Lys-His) of\nDONepi and the canonical catalytic triad (Ser/Thr-Tyr-Lys) of SDR-type epimerases are highlighted\nwith blue and red backgrounds, respectively. Aligned sequences and their corresponding PDB\naccession numbers are listed as follows: UDP-GlcA C4-epimerase (Bacillus cereus, 6ZLA); UDP-\nGlcA C4-epimerase (Streptomyces viridosporus, 6KV9); UDP-Gal C4-epimerase (Escherichia coli,\n1UDA). b, Structural comparison of the active sites between DONepi and representative SDR-type\nepimerases. Amino acid residues and their positions corresponding to the active sites are labeled\naccordingly. The C4 position of nicotinamide coenzyme molecules is indicated.\n(2) A second issue concerns the catalytic performance of DONepi itself. The enzyme\nexhibits a low turnover rate (kcat = 0.3 min⁻¹), which substantially limits its practical\nbiotechnological utility. Other reported enzymes capable of catalyzing the same\nreaction display significantly higher turnover rates, such as DepA (1.69 s⁻¹; Toxins 2024\nMar 5;16(3):136) and DepB 3 s-1 (Sci Rep. 2022 Aug 30;12:14737). This comparison\nhighlights that the enzyme, in its current form, is markedly less efficient than existing\nalternatives.\nResponse: Thank you for your insightful comment. The enzymatic systems consisting\nof two distinct proteins—pyrroloquinoline quinone (PQQ)-dependent alcohol\ndehydrogenases (e.g., DepA) and NADPH-dependent aldo-keto reductases (e.g.,\nDepB)—indeed exhibit high epimerization activity toward DON. Our laboratory has\nalso been investigating this dual-protein system for several years (He et al., Food Chem.\n2020, 321: 126703). However, as mentioned in the Introduction section of the\nmanuscript (Lines 45-48), a key limitation of such epimerization detoxification systems\nis their dependence on the cofactor PQQ, which is essential for the PQQ-dependent\nalcohol dehydrogenases that catalyze the first step of DON epimerization. Because\nPQQ cannot be naturally synthesized by industrially relevant strains (e.g., yeast, E. coli),\nthe heterologously expressed PQQ-dependent alcohol dehydrogenases show no\ncatalytic activity in these hosts if no exogenous PQQ is added. Moreover, the\nexogenous addition of PQQ leads to prohibitively high production costs, which severely\nrestricts the practical application of this enzymatic system. Thus, PQQ-independent\nDON epimerases are in urgent demand for overcoming these limitations.\nIn contrast, DONepi, the enzyme identified in our study, relies solely on NADP⁺ as a\ncofactor. Critically, this cofactor is naturally present and metabolically accessible in the\naforementioned industrial chassis strains and other biological systems, eliminating the\nneed for exogenous supplementation of costly cofactors such as PQQ. Thus, DONepi\nrepresents a promising alternative for DON epimerization by resolving the cofactor-\nrelated bottleneck associated with the dual-protein system. Our results have\ndemonstrated that heterologous expression of the single DONepi gene significantly\nenhanced DON tolerance in transgenic Arabidopsis thaliana (Supplementary Fig.\n36), validating its potential for practical biotechnological applications. Regarding the\nreviewer’s concern about the relatively low catalytic performance of DONepi, rational\nprotein engineering strategies, particularly the rapidly advancing AI-guided design\napproach, will be employed in future work to improve its catalytic efficiency by\nleveraging our experimentally determined protein structure and elucidated catalytic\nmechanism. This strategy has been successfully applied to enhance the catalytic\nefficiency of numerous enzymes (Lee et al., Adv. Sci., 2025, 12: e02914; Yang et al.,\nACS Catal., 2025, 15: 11664-11672; Fei et al., Cell, 2025, 188: 1-19). We firmly believe\nthat our study provides a solid foundation for global researchers to identify more\nefficient DONepi homologs and improve their catalytic efficiency. Beyond this single-\nenzyme optimization, our future research will further focus on characterizing the\ncomplete DON degradation pathway, with subsequent co-expression of DONepi and\nall other enzymes involved in this pathway in engineered strains (e.g., yeast, E. coli).\nThis will thereby enable the development of robust biocatalytic systems for the\ncomplete detoxification of DON.\nWe hope these explanations, supporting references, and future research plans\nadequately address your concern."}, {"round": 4, "reviewer_comments": {"reviewer_1": "Reviewer concerns have been addressed."}, "author_response": ""}]

Decision letter