Molecular mechanism underlying regulation of chalcone synthase by chalcone isomerase-like protein

Domain: biology
Published: 2026-03-14
Editorial outcome: major_revision

Abstract
Flavonoids are essential for plant growth and environmental adaptation. Chalcone synthase (CHS) directs metabolic flux into flavonoid biosynthesis, but its catalytic promiscuity limits the biosynthetic efficiency. Although the chalcone isomerase-like protein (CHIL) has been characterized as physically interacting with and regulating CHS, the underlying mechanism remains elusive. Here, we report the crystal structure of the CHS-CHIL complex, revealing that CHIL modulates CHS function by gating the substrate-binding pocket entrance through its β-hairpin region. Molecular dynamics simulations indicate that regulation occurs early in the catalytic cycle, affecting substrate binding or early intermediate formation. CHIL enhances CHS activity and product specificity by promoting CoA release and stabilizing key amino acid residues. His36 within the β-hairpin is functionally critical; its substitution with leucine in Arabidopsis and other plant species markedly improves CHS catalytic efficiency and specificity. This regulatory mechanism is evolutionarily conserved across land plants, from bryophytes to angiosperms.

Guided by evolutionary analysis, we engineer a CHIL variant (H36E/F37T) that significantly enhances CHS activity. Collectively, our findings establish a conserved binding-conformational regulation paradigm that governs metabolic flux into the flavonoid biosynthetic pathway and provides practical strategies for enhancing flavonoid production and composition in crops.

Introduction
Flavonoids represent a diverse and fascinating polyphenolic group of plant specialized metabolites with widespread and significant implications for human health, such as anti-aging, anti-inflammatory and reducing the risk of cardiovascular diseases1-4. They are ubiquitously distributed in land plants and play critical roles in plant growth, development, and defense mechanisms against pathogens and environmental stresses1,5,6. Flavonoids contain three rings (C6-C3-C6) as the basic skeleton, and their biosynthesis comes from the general phenylpropanoid pathway1,3,7. Through a sequential cascade of enzymatic reactions catalyzed by key enzymes like chalcone synthase and chalcone isomerase, the fundamental metabolic precursor L-Phenylalanine is converted into structurally diverse flavonoid subclasses, as illustrated in Fig. 1a.

Chalcone synthase (CHS) stands as a pivotal enzyme in the flavonoid biosynthetic pathway of plants, catalyzing the initial committed step that initiates the synthesis of a wide-ranging assortment flavonoid compounds7-9. Belonging to the type III polyketide synthase (PKS) enzyme family, CHSs have been isolated and their structures elucidated from numerous plant species, including alfalfa (Medicago sativa)8, fern species (Odontosoria chinensis, Lindsaea orbiculata)10,11 and Arabidopsis thaliana12. Functioning as a homodimer, CHS possesses two independent active pockets that facilitate a series of decarboxylation, condensation, and cyclization reactions, ultimately generating the production of naringenin chalcone (NC) from one p-coumaroyl-CoA and three malonyl-CoA molecules10-12 (Fig. 1a). Each CHS monomer is composed of two structural domains: an upper domain that houses the active site with key catalytic amino acids, and a lower domain that provides the necessary spatial configuration for the formation of the tetraketide intermediate required for chalcone formation13. Notably, the Cys-His-Asn catalytic triad and  pseudo symmetric motif is highly conserved in plant CHSs10-12.

Like other enzymes involved in plant specialized metabolism, CHS also generates side products during its catalytic reactions14-20. CHS catalyzes the formation of naringenin chalcone (NC) as the main product, alongside the derailment side product 4-coumaroyltriacetic acid lactone (CTAL) (Fig. 1a)21,22. This phenomenon, known as "catalytic promiscuity", is prevalent in plants and serves as a fundamental mechanism for environmental adaptation and adaptive evolution21. However, it reduces the efficiency of flavonoid biosynthesis in plants and poses a significant challenge for leveraging CHS in synthetic biology approaches, whether in plant-based systems or microbial platforms, for the production of specific flavonoid compounds with potential health benefits. Interestingly, previous studies have demonstrated that the chalcone isomerase-like protein (CHIL) plays an important role in regulating CHS catalytic promiscuity through protein-protein interactions21,22.

Chalcone isomerase (CHI) is the second committed enzyme in the flavonoid biosynthetic pathway, which catalyzes the intramolecular and stereospecific cyclization of naringenin chalcone to form naringenin22,23. Phylogenetic analysis indicates that CHI is exclusively found in vascular plants, and CHI-fold proteins can be classified into four distinct types22,24. Type I or type II CHIs exhibit catalytic activity, which isomerize naringenin chalcone to form (2S)-naringenin, or catalyze naringenin chalcone and isoliquiritigenin (6'-deoxychalcone) to generate (2S)-naringenin and liquiritigenin (5-hydroxyflavanone), respectively; type III CHIs, which can bind fatty acids and are believed to be associated with fatty acid metabolism, lack any detectable CHI catalytic activity; similarly, type IV CHIs, also known as CHILs (chalcone isomerase-like proteins), have mutated several key amino acid residues essential for catalytic function, resulting in a complete loss of CHI catalytic activity22,24,25. A loss-of-function mutation in CHIL was found to decrease accumulation of flavonoids and anthocyanins in Ipomoea nil26. Similar results were observed in Arabidopsis thaliana, where overexpression of CHIL partially rescued the phenotype by enhancing flavonoids production27. A proposed mechanism suggests that CHIL physically interacts with CHS altering its catalytic promiscuity and subsequently improving the efficiency of flavonoid biosynthesis21,22,28,29. Despite the identification of CHILs across various land plant species and the characterization of their conserved function, the precise molecular mechanisms underlying their activity remain elusive.

In this study, we found that the expression patterns of CHS and CHIL were synergistically coordinated. Subsequently, the CHS-CHIL complex was successfully expressed and purified, and its crystal structure was determined. Integration of structural and biochemical analyses revealed that CHIL binds to CHS through an extensive interface, modulating the enzyme's product specificity. And a notable conformational change occurred in the β-hairpin region of CHIL. Furthermore, the H36L mutant significantly enhanced CHIL's regulatory function on CHS, demonstrating potential for enhancing the nutritional value of crops. Enzymatic assays confirmed that CHIL proteins from five major lineages of land plants consistently enhanced the activity and product specificity of their cognate CHSs, demonstrating functional conservation across land plant evolution.

Results
Regulation the activity and product specificity of CHS by CHIL

Before investigating the structure and function of the CHS-CHIL complex, we first analyzed the specific cell types and developmental stages in which the CHS and CHIL proteins are expressed and exert their functions. We analyzed single-nucleus RNA sequencing (snRNA-seq) data from a previous study encompassing six Arabidopsis leaf developmental stages (S1–S6)30. The results revealed that genes within the flavonoid biosynthesis pathway were significantly enriched at the mature S3 stage (Supplementary Fig. 1a-d). Analysis of cluster 4 showed preferential expression of CHS (AT5G13930) and CHIL (AT5G05270) in C4 cells (Supplementary Fig. 1e), with enrichment specifically observed in epidermal cells at stages S1, S2, S3, and S6 (Supplementary Fig. 2). Correlation analysis identified stage- and cell-type-specific co-expression patterns between CHS and CHIL (Supplementary Fig. 1f), suggesting that CHS and its regulator CHIL act synergistically to regulate the flavonoid metabolic pathway. The significant correlation between CHS and CHIL in the epidermal cells of Arabidopsis leaves indicates their involvement in flavonoid biosynthesis, which may assist the leaves in resisting adverse environmental stresses such as ultraviolet radiation.

Subsequently, to investigate the role of noncatalytic CHIL in determining the product specificity of CHS, we cloned and expressed the CHS and CHIL proteins of Arabidopsis thaliana (Supplementary Fig. 3). The purified proteins were performed the in vitro enzymatic assays, and the reaction products were analyzed by HPLC-MS. Two prominent product peaks (P1 and P2) were observed during the HPLC-MS analysis when CHS was assayed with p-coumaroyl-CoA and malonyl-CoA as substrates, which is consistent with previous studies (Fig. 1b)10,11. Through comparison with authentic standards and tandem MS/MS analysis, P1 and P2 were identified as the major product of (2S)-naringenin (an isomer of NC that forms via a non-enzymatic isomerization) and the byproduct CTAL, respectively (Fig. 1b, Supplementary Fig. 4). Notably, compared to assays containing only CHS, the addition of CHIL significantly altered the product profile: the proportion of CTAL decreased from 27.5% to 15.9% of total products, while the production of (2S)-naringenin increased approximately 1.5-fold (Fig. 1b, Supplementary Fig. 5). These results support previous proposals21,22, and strongly indicate that CHIL functions as a specialized auxiliary protein, largely increasing the yield of the main product to modulate CHS product specificity.

Since CHIL belongs to the CHI family, could catalytically active CHI also interact with CHS in a similar manner to regulate its enzymatic activity and product specificity? To analyze the functional differences between CHI and CHIL, we performed pull-down assays with CHS using each protein individually. The results demonstrated that CHIL bound to CHS, whereas CHI did not (Fig. 1c). Furthermore, co-expression and purification of CHS and CHIL by gel filtration confirmed their stable interaction and complex formation (Fig. 1d). Subsequently, purified CHI and CHIL were individually incubated with CHS, and their effects were assessed by enzymatic assays. The results showed that CHIL dramatically enhanced both the activity and product specificity of CHS. In contrast, CHI only slightly increased CHS activity, potentially attributable to CHI utilizing the CHS product NC as its substrate, thereby indirectly increasing metabolic flux through the pathway (Fig. 1e). Phylogenetic analysis also confirmed that CHI and CHIL form distinct clades despite sharing the conserved CHI-fold domain24. These findings demonstrate that CHI and CHIL have undergone distinct functional differentiation during evolution.

The overall structure of CHS-CHIL complex

To elucidate the structural basis for CHIL's specific binding to CHS (unlike CHI) and its consequent regulation of CHS activity and product specificity, we determined the crystal structure of the CHS-CHIL heteromeric complex. Through a two-step purification strategy, the CHS-CHIL complex of A. thaliana was purified, crystallized, and condition optimized. Its crystal structure was successfully determined at a resolution of 1.9 Å using the molecular replacement method (Fig. 2, Supplementary Fig. 6, Supplementary Table 1). The crystal structure of the CHS-CHIL complex reveals a heterotetrameric assembly within the asymmetric unit, comprising two CHS and two CHIL subunits. This arrangement adopts a distinctive "blooming flower" architecture when viewed along its central axis. At the structural core, two CHS monomers form a symmetric homodimer, with each flanking CHIL subunit bound to an individual CHS monomer. Notably, the two CHIL subunits lack direct intermolecular contacts (Fig. 2a, b).

The CHS structure within the CHS-CHIL complex retains the characteristic  thiolase fold8,12, consistent with functional conservation among CHS orthologs. To investigate structural changes induced by complex formation, we superimposed CHS homodimers from A. thaliana (AtCHS, PDB ID code 6DXB [https://doi.org/10.2210/pdb6DXB/pdb]) and Cyclosorus parasiticus (CpCHS, PDB ID code 7VF0 [https://doi.org/10.2210/pdb7VF0/pdb]) with the complexed CHS, whose root-mean-square deviation (RMSD) are 0.219 Å and 0.375 Å for the C atoms, respectively. The results revealed that the overall structure of CHS in CHS-CHIL complex closely resembled AtCHS and CpCHS, including the NAR and CoA binding pockets. However, subtle conformational differences were observed in the loop regions at the top of the upper domain (Fig. 2c).

Similarly, structural comparison of free AtCHIL (PDB ID: 4DOK [https://doi.org/10.2210/pdb4DOK/pdb]) with complex-bound CHIL revealed high conformational conservation with RMSD values of 0.352 Å for the C atoms (Fig. 2d). The CHIL component of CHS-CHIL complex possesses nine -strands and seven -helices, forming the characteristic CHI-fold domain conserved across the CHI protein family23,24. While the global CHIL architecture remains largely unaltered upon CHS binding, a significant conformational change occurs in its -hairpin region. This conformationally remodeled region inserts into the substrate binding pocket entrance of CHS, potentially modulating substrate access or product release (Fig. 2d).

The interface of CHS and CHIL in the complex

A detailed analysis of the interaction surface between CHS and CHIL was further conducted. The interaction surface is formed by -helices and loops from the two CHS molecules and one CHIL molecule, burying a total surface area of 2,758 Å2 (Fig. 3a-c). As underscored in Fig. 3a to 3c, the CHS-CHIL binding interface is formed by two principal contact areas, ITF1 (Interface 1) and ITF2 (Interface 2), collectively creating an inverted "L" shape. The ITF1 contains residues Ile33, Ile35, His36, Phe37, Leu38, Val40, and Phe42 from CHIL, which mainly form hydrophobic interactions with residues Leu57, Lys60, Arg63, Met64, Lys67, Leu211, Leu214, Val215, Ala218, Leu219, Leu273, Leu274, Pro278, Pro313, and Ala314 from CHS (Fig. 3b). These residues from CHS form a hydrophobic pocket, functioning like a “lock”, while the -hairpin of CHIL protrudes into the hydrophobic pocket, acting as a “key” to form a flexible switch. The ITF2 contains residues Lys94, Lys97, Gln100, Gln104, Asn107, and Tyr138 from CHIL, which form hydrogen bonds with residues Asp212, His272, Asp101, Asp261, His272, Lys275, Asp257, and Arg265 from CHS, respectively (Fig. 3c). Additionally, the residues Arg110 of CHIL and Lys151, Tyr147, Asp141 of CHS form hydrogen bonds with residues Asn107 of CHIL and Asp257, His272 of CHS, respectively, to dictate their correct positioning. The side chain of residues Ile96 forms hydrophobic interactions with Lys94 and Gln100 of CHIL, to assist in orientating these residues correctly. Notably, the lower part of ITF2 exhibits greater interaction stability than the upper part, suggesting that the lower part may provide a dynamic hinged pivot that allows the upper part to move more flexibly.

To verify the significance of the interaction surfaces involved in CHS-CHIL, we performed mutation-based functional analyses. In vitro pull-down experiments showed that the CHS mutations D212A, D261A, H263A, H272A, and K275A in ITF2 significantly reduced the interactions between the CHS and the CHIL (Fig. 3d, Supplementary Fig. 7a). Accordingly, these mutations also severely affected the activity of both the CHS-CHIL complex and isolated CHS (Fig. 3e). These key ITF2 residues are adjacent to the active pocket (Supplementary Fig. 8a, b). Such mutations may impair CHS function by affecting substrate binding or catalytic activity. Therefore, observed effects in CHS mutants may not solely reflect disrupted CHS-CHIL interactions. Thus, CHIL mutants targeting ITF2 were constructed and analyzed by pull-down and activity assays. The interactions between CHS and CHIL were slightly decreased by K94A mutations, and severely impaired by K97A, Q100A, Q104A, Y138A, and M3A (K94A/K97A/Q100A) (Fig. 3f, Supplementary Fig. 7b). Consistently, K94A minimally affected enzymatic activity, whereas other single mutations severely impaired it. The multiple mutations M5A (K94A/K97A/Q100A/Q104A/N107A) and M6A (K94A/K97A/Q100A/Q104A/N107A/Y138A) completely abolished the regulatory function of CHIL (Fig. 3g). Similarly, the key amino acid residues in ITF1, located within and around the -hairpin of CHIL, were selected for mutational analyses. As shown in Fig. 3h and 3i, the interactions between CHS and CHIL were moderately impaired by H36A but severely impaired by F37A and M4A (I35A/H36A/F37A/L38A) mutant (Supplementary Fig. 7c). Furthermore, we performed ITC experiments on several representative mutants of the CHIL protein from ITF1 and ITF2 (Supplementary Fig. 9). The results showed that the binding affinity between CHIL and CHS was 7.8 M. Compared to wild-type CHIL, the H36A mutant still bound to CHS, but with weakened binding affinity, as reflected by a 50% increase in the KD value. In contrast, no detectable binding was observed for the F37A, M4A, K97A, Q100A, or M3A mutants of CHIL, as no reliable KD values could be determined. Thus, these mutations (from ITF1 and ITF2) almost eliminated the beneficial effects of CHIL on CHS. Collectively, these results suggested that both ITF1 and ITF2 mediate the biological functions of CHIL, confirming that CHIL regulates CHS activity and product specificity through binding modulation.

Site 36 is central to the regulatory function of CHIL

In the structure of CHS-CHIL complex, we observed the -hairpin region of CHIL protruding into the catalytic pocket entrance of CHS, while also forming the vital interaction interface ITF1 (Fig. 2, Fig. 3a, b, Supplementary Fig. 8b). This structural arrangement suggests that the -hairpin region of CHIL may play a regulatory role in CHS function. Thus, the reaction products of NAR and CoA were modeled into the CHS-CHIL complex from CpCHS (PDB ID code 7VF0 [https://doi.org/10.2210/pdb7VF0/pdb]), which revealed the significant steric hindrance between the CHIL's -hairpin and CoA (Fig. 4a, b). To investigate this phenomenon, we co-purified the GST-tagged CHS with 6×His-tagged CHIL using Ni2+-NTA affinity chromatography, then incubated the column with CoA for 30 minutes. After washing with ten column volumes of lysis buffer, a substantial amount of CHS was eluted, resulting in a sub-stoichiometric CHS:CHIL ratio (Fig. 4c). This biochemical evidence confirms an antagonistic relationship between CoA and CHIL, explaining our failure to obtain crystals of the CHS-CHIL complex with bound NAR and CoA despite several attempts. These findings suggested that CHIL modulates the activity and product specificity of CHS by accelerating CoA release to increase reaction rates.

To further investigate the role of β-hairpin region in CHIL, we first analyzed the conservation of amino acid residues in this area. As shown in Fig. 4d, the residues from Glu34 to Phe37 are highly conserved in plants (Fig. 4d). Structural analysis of the CHS-CHIL complex revealed that residue His36 of CHIL influences the position and orientation of residue Phe220 through hydrophobic interactions with residues Val215 and Leu219 of CHS (Fig. 4e). Notably, Val215, Leu219, and Phe220 in CHS are also evolutionarily conserved in plants (Fig. 4d). Phe220 is defined as one of the key residues to form the active site of CHS, providing van der Waals interactions for substrate binding and participating the catalytic process8. The mutations of V215A/L219A and F220A abolish CHS activity, similar to the F271A mutation. Previous studies have shown that Phe271 separates the coumaroyl-binding site from the cyclization pocket and may function as a mobile steric gate during successive rounds of polyketide elongation, underscoring its essential role in CHS catalysis8. To assess the function of position 36 in CHIL, His36 was replaced with different types of amino acid residues to evaluate its role, which possessed different sizes and charges of the side chain. Replacing it with valine (H36V), charged residues (H36D and H36E), or polar residues (H36Y and H36N) severely impaired the CHIL function, whereas substitution with hydrophobic amino acid residues (H36I, H36F and H36W) resulted in product distributions similar to those of the CHIL wild-type (Fig. 4f). Strikingly, substituting methionine or leucine for histidine (H36M or H36L) increased naringenin yield by 39% or 79%, respectively, with its proportion in the total products also rising from 88% to 91% or 95% (Fig. 4f).

Sequence alignment and structure analysis unequivocally demonstrated that CHIL exhibits extraordinary conservation throughout plant evolution. Based on this conservation, we investigated whether the wild-type CHIL protein of Arabidopsis thaliana and its functional-enhanced H36L mutant could regulate the activity of CHS in heterologous species. Functional characterization revealed that CHIL from Arabidopsis thaliana significantly enhanced both the catalytic activity and product specificity of CHSs from rice, maize, and soybean (Fig. 4g). Notably, the H36L mutant further potentiated CHIL’s regulatory capacity in a species-independent manner (Fig. 4g). Furthermore, we generated H38L and H41L mutants at the corresponding sites in ginkgo and rice CHIL, respectively. Functional assays revealed that these mutants elicited significant enhancements in CHIL-mediated CHS regulation, with activity improvements of 14% and 20% observed in ginkgo and rice, respectively (Fig. 4h). Collectively, our findings demonstrate that the H36 residue is critical for CHIL-mediated regulation of CHS activity. The H36L mutation confers a significant enhancement to wild-type CHIL function across diverse plant species, confirming its conserved regulatory role in chalcone biosynthesis.

Taken together, CHIL regulates CHS activity through the contact surface ITF1 and ITF2 (Fig. 3 and 4, Supplementary Fig. 8a-b). Specifically, via its β-hairpin domain, CHIL enhances the enzymatic activity and product specificity of CHS by facilitating CoA release and aiding in the positioning of key amino acid residues within CHS (Fig. 3 and 4, Supplementary Fig. 8b). Based on these structural and biochemical analyses, the presence of CHIL should enhance the catalytic efficiency of CHS by increasing the kcat values for both substrates. Given this, a more detailed analysis of the enzyme kinetics was conducted. For malonyl-CoA, the enzymatic kinetic parameters were characterized for individual CHS, as well as for CHS in combination with wild-type CHIL and representative CHIL mutants (Supplementary Table 2). The results demonstrate that CHIL addition markedly enhanced CHS catalytic efficiency, increasing both Vmax and kcat by approximately 80%, while also raising the Km value by about 53%. Kinetic characterization of CHIL mutants further revealed distinct functional effects: the H36L mutant, which enhances CHS activation compared to wild-type CHIL, elevated Vmax and kcat by an additional 36%, indicating a strengthened regulatory capacity. In contrast, two binding-deficient mutants, M4A located at ITF1 and Q100A located at ITF2, exhibited kinetic parameters similar to those measured in the absence of CHIL. This supports the conclusion that the CHIL-CHS interaction is essential for the observed catalytic enhancement. Similarly, for p-coumaroyl-CoA, CHIL addition significantly improved CHS catalytic efficiency, with Vmax and kcat increasing by approximately 50% and Km rising by about 65%. The H36L mutant further enhanced this effect, elevating Vmax and kcat by an additional 66% relative to wild-type CHIL. The enzyme activity assays confirmed that CHIL significantly enhances the catalytic activity of CHS. This finding is consistent with previous studies reporting a 2- to 15-fold increase in kcat in the presence of CHIL across diverse plant species21.

Evolution of CHIL

To further investigate the role of critical residues in the evolutionary relationship between CHIL and CHI, we compared their differences within the activity pocket and at the CHS contact surface (Supplementary Fig. 10). Superposition analysis revealed that Trp184 and Tyr185 in CHIL, with their bulky side chains, occupy the same active site positions as in MsCHI (PDB ID code 1EYQ [https://doi.org/10.2210/pdb1EYQ/pdb]) and AtCHI (PDB ID code 4DOI [https://doi.org/10.2210/pdb4DOI/pdb]) (Supplementary Fig. 10a-b). Other residues that form the substrate-binding pocket are identical between CHIL and MsCHI, except for Thr31/Arg36, Asp32/Gly37, Ile33/Leu38, Val90/Gly95, Val92/Lys97, Ile96/Leu101, Gln100/Glu105, Gln104/Lys109, Leu105/Val110, and Thr108/Asn113 (Supplementary Fig. 10a). Furthermore, we analyzed the CHS-CHIL complex interface residues on CHIL and compared them with AtCHI. Within ITF1, several residues were conserved, except for Ile33/Leu49, His36/Gln52, Phe37/Gly53, and Leu38/Lys54 (Supplementary Fig. 10c). In contrast, within ITF2, only Gln100/Gln104 remained identical (Supplementary Fig. 10d).

To comprehensively elucidate the evolutionary trajectory of CHIL proteins in plants, we selected 18 representative species spanning diverse phylogenetic lineages for analysis, including a cyanobacterium (Anabaena cylindrica), a green alga (Chlamydomonas reinhardtii), a liverwort (Marchantia polymorpha), a moss (Physcomitrella patens), two lycophytes (Selaginella martensii and Selaginella kraussiana), three ferns (Azolla filiculoides, Odontosoria chinensis and Lindsaea orbiculata), two gymnosperms (Ginkgo biloba and Pinus tabuliformis), and seven angiosperms. The angiosperms comprised a basal angiosperm (Amborella trichopoda), three eudicots (Arabidopsis thaliana, Solanum lycopersicum, Glycine max), and three monocots (Oryza sativa, Zea mays, Triticum aestivum). Using the Arabidopsis thaliana CHIL sequence, we performed BLASTP searches against protein databases, identifying 7,240 candidate genes predominantly from the Magnoliopsida class (Supplementary Table 3). To ensure representativeness, candidate genes from these 18 species listed above were selected for subsequent analysis. Leveraging residues involved in the CHI active site and the CHS-CHIL complex interface as discriminatory criteria, we developed a Structure-based Key Amino acid residues clustering plus Sequence Alignment method (SKSA, see methods for details) (Fig. 5a, and Supplementary Fig. 11). Ultimately, 11 CHIL proteins from 10 plant species were successfully expressed in E. coli and stably purified, which served as the foundation for constructing a phylogenetic tree (Fig. 5a, Supplementary Fig. 13). Employing the same strategy, the evolutionary tree for CHI proteins was also constructed (Supplementary Table 3, Supplementary Fig. 12a). The results suggested that the CHIL and CHI proteins originated in bryophytes and have been conserved across land plant species, encompassing lycophytes, ferns, gymnosperms, and angiosperms.

To validate the functional roles of the cloned CHILs, 11 CHS candidates were obtained from the corresponding 10 plant species using the same strategy employed for searching and cloning CHILs. Similarly, a phylogenetic tree was constructed based on these CHS protein sequences (Supplementary Fig. 12b, Supplementary Fig. 14). Comparison of the critical residues mentioned above revealed that most are highly conserved. Enzymatic assays of CHIL with corresponding CHS from various plant species demonstrated that CHIL proteins from distinct evolutionary lineages consistently modulate the product specificity and enhanced the catalytic activity of CHS (Fig. 5b). Notably, the production of (2S)-naringenin increased 2.4- to 5-fold in gymnosperms and angiosperms, but dramatically increased between 6- to 14-fold in bryophytes, lycophytes, and ferns (Supplementary Fig. 12c). These results further indicate that the product specificity and enzyme activity of CHS in bryophytes, lycophytes, and ferns are generally lower than in gymnosperms and angiosperms.

Furthermore, we analyzed the interaction interfaces ITF1 and ITF2, and the results revealed differences in the residues at sites 36 and 37 of the CHIL -hairpin region between euphyllophytes (ferns, gymnosperms and angiosperm) and non-euphyllophytes (bryophytes and lycophytes). Specifically, the glutamate (E) and threonine (T) present at sites 36 and 37 in non-euphyllophytes have been substituted with histidine (H) and phenylalanine/tyrosine (F/Y) in euphyllophytes. Site-directed mutants H36E and H36E/F37T of CHIL were constructed, and the enzymatic assays demonstrated that the H36E/F37T double mutant significantly enhanced enzymatic activity by approximately 16% (Fig. 4f). These findings not only clarify the evolutionary divergence of CHIL functional sites but also provide a foundation for designing crops with high flavonoid content based on evolutionary history.

Collectively, our findings provide compelling evidence that the phenylpropanoid metabolic pathway likely emerged as a crucial specialized metabolic adaptation in land plants following their transition from aquatic to terrestrial habitats. This pathway enabled them to cope with environmental challenges such as UV-B radiation, drought, and interactions with pathogens and herbivores.

Methods
Gene Cloning and Protein Purification

Total RNA was extracted with Trizol reagent (Invitrogen, Cat No./ID: 217084). Total RNA (1 µg) was treated with 1 µL of DNase I (1.0 unit/μL; Thermo Fisher, Cat No./ID: EN0521) and complementary DNAs (cDNAs) were synthesized with the All-in-one Kit (Vazyme, Cat No./ID: R333-01). To construct plasmids for prokaryotic expression of full-length CHS (residues 1-395), CHIL (residues 1-209) and CHI (residues 1-246) of Arabidopsis thaliana in Escherichia coli, the coding sequence of these genes were PCR-amplified and cloned into pETDuet-1 vector with a 6×His-tag at the N-terminus. The constructs for point mutations were generated by overlap PCR and verified by DNA sequencing. All the plasmids were transformed into the E. coli BL21 (DE3) strain or Rosetta (DE3) cells. The transformed bacterial cells were grown in Luria-Bertani (LB) medium supplemented with ampicillin at 37°C to an OD600 of 0.8, and then induced by 0.2 mM isopropyl β-D-thiogalactopyranoside (IPTG, Sangon Biotech, Cat No./ID: A100487) for 12 h at 18 °C. The cells were harvested and resuspended in buffer A (20mM Tris-HCl, pH 8.0, 100mM NaCl) supplemented with 1mM phenylmethane sulfonyl fluoride (PMSF, Sangon Biotech, Cat No./ID: A100754). Cells were lysed by a high-pressure cell disruptor at 15,000 p.s.i. (pounds per square inch), and the lysate was centrifuged at 20,000 × g for 45 min. The supernatant was loaded onto a Ni2+-NTA affinity column (Qiagen, Cat No./ID: 30230) and washed with buffer A plus 20mM imidazole. Proteins were eluted by buffer A plus 250mM imidazole and purified by gel filtration using a Superdex 200 Increase column (Cytiva, Cat No./ID: 28990944) in buffer A (20mM Tris-HCl, pH 8.0, 100mM NaCl) on an ÄKTA pure system. The sample was eluted at a flow rate of 0.4 mL/min. The elution profile was monitored by measuring the absorbance at 280 nm. Peak fractions were collected and concentrated for subsequent structural and biochemical studies.

Crystallization, Data Collection and Structure Determination

Protein was concentrated to 10 mg/mL for crystal screening, and crystals of the CHS-CHIL complex were grown at 20°C using the sitting-drop vapor diffusion method by mixing 0.5 μL of protein with 0.5 μL of reservoir solution containing 0.2 M trimethylamine N-oxide dihydrate, 0.1 M Tris pH 8.5, 20% (w/v) polyethylene glycol monomethyl ether 2000. All crystals were transferred into cryoprotectant solution containing their respective mother liquor plus 30% (v/v) glycerol, before being flash frozen in liquid nitrogen for storage. Data collection was performed at BL19U1/ BL18U/ BL02U beamline of the Shanghai Synchrotron Radiation Facility (SSRF) under 100 K liquid nitrogen stream (wavelength = 0.9798 Å). The data were processed with HKL300046, and the initial phase was determined by molecular replacement. All of the models were manually built in Coot47, and were refined by iterative rounds of manual adjustment with Coot and refinement with Phenix48. Data collection and structure refinement statistics are shown in Supplementary Table 1.

In Vitro Enzymatic Assay

The enzymatic assay process and HPLC parameters were applied according to the previously reported studies 10,11,22. Specifically, the CHS and CHS-CHIL complex enzymatic assays mixture, including 50 μM p-coumaroyl-CoA (Shanghai YuanYe Bio-Technology, Cat No./ID: S25276), 100 μM malonyl-CoA (Sigma-Aldrich, Cat No./ID: M4263), and 2.5 μg of each purified proteins in 20 mM Tris HCl buffer at pH 7.0 reached the final volume of 100 μL for 10 min at 35°C. The reactions terminated by the addition of 10 μL of 10% glacial acetic acid and 200 μL methanol. After centrifugation, a 10 μL volume of the supernatant was separated through a reverse-phase C18 column (Agilent® ZORBX SB-C18, 5 m 4.6×150 mm) using an Agilent 1260 high-performance liquid chromatography (HPLC) coupled with a 6460 Triple Quadrupole LC-MS system. Samples were analyzed by electrospray ionization (ESI) in negative ionization mode. The column temperature was maintained at 30°C, and a flow rate of 1.0 ml/min was used. A binary gradient system was employed, consisted of solvent A (methanol) and solvent B (water plus 0.1% formic acid). A linear gradient of 30% A and 70% B to 80% A and 20% B was supplied over 20 min. Standard naringenin (Shanghai YuanYe Bio-Technology, Cat No./ID: B21596) solutions of reference compounds were used for calibration.

For kinetic characterization, assay mixtures contained 2.5 g of purified CHS and 2.5 g of purified CHIL (either wild-type or mutants) with varied substrate concentrations (5-100 M). Kinetic parameters for p-coumaroyl-CoA were determined in the presence of 100 M malonyl-CoA; similarly, malonyl-CoA kinetics were assessed with 50 M p-coumaroyl-CoA. Reactions proceeded for 10 min at 35°C and were terminated by adding 10 L of 10% glacial acetic acid and 200 L methanol. Product analysis was performed as described above. Kinetic values were derived by nonlinear regression to the Michaelis-Menten equation using GraphPad Prism 10.

In Vitro Pull-Down Assays

The wild-type and mutated versions of CHS, CHIL and CHI were generated by PCR-based mutagenesis. The oligonucleotide primers are given in Supplementary Table 4. The 6×His-tagged wild-type and mutated CHIL or CHI proteins were co-expressed with Glutathione S-transferase (GST)-tagged wild-type and mutated CHS in E. coli Rosetta (DE3). After cell disruption and centrifugation, the supernatants were loaded to Ni2+-NTA affinity resin (Qiagen, Cat No./ID: 30230). The nonspecific bound protein was washed off. The resin was boiled, examined with Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and visualized by Coomassie Blue staining.

ITC Analysis

ITC experiments were performed with the MicroCal ITC200 system (Malvern Instruments) at 20°C in PBS buffer (Biopico Life Sciences, Cat No./ID: 830101). Proteins were purified as described above, using PBS as the buffer; For detection of CHIL and its mutants binding to CHS, the syringe was filled with 0.5 mM CHIL or its mutants, and the cell was filled with 50 M CHS protein. The sample in the syringe was added to the sample cell by sequential injections of 2 L aliquots followed by 120 s of equilibration after each injection. There was a total of 20 injections. For analysis, the heat released by each injection was integrated, and the background was subtracted. The data were fit to the Wiseman isotherm with the Origin ITC analysis package. The experiments were repeated at least three times for each sample.

Phylogenetic Tree Construction

To investigate the evolutionary relationships of CHS, CHILS, and CHI proteins, we performed separate BLASTP searches using Arabidopsis thaliana CHS (AT5G13930.1), CHIL (AT5G05270.1), and CHI (AT3G55120.1) sequences against protein databases of other selected species. For each of the three protein families, a separate phylogenetic tree was constructed to identify the closest homologs across species based on phylogenetic proximity. Selected sequences included: Marchantia polymorpha (LCHIL: PTQ28039; CHI: PTQ28320; CHS: PTQ44319), Physcomitrella patens (CHIL: Pp6c4_13620V6.3; CHI: Pp6c26_2490V6.3; CHS: Pp6c24_6740V6.1), Selaginella kraussiana (CHIL: SK08G12910.mRNA1; CHI: SK06G16750.mRNA1A; CHS: SK02G19610.mRNA1), Selaginella martensii (CHIL: Sm_0626930.1; CHI: Sm_0310740.1; CHS: Sm_0158520.1), Azolla filiculoides (CHIL: Azfi_s0242.g059817; CHI: Azfi_s0030.g024403; CHS: Azfi_s0168.g054633), Ginkgo biloba (CHIL: evm.model.chr1.2875; CHI: evm.model.chr4.1966; CHS: evm.model.chr6.1424), Pinus tabuliformis (CHIL: Pt5G49490.1; CHI: PtQG07910.3; CHS: Pt3G28890.1), Amborella trichopoda (CHIL: XP_011622657.1; CHI: XP_006850685.1; CHS: XP_011627026.1), Odontosoria chinensis (CHIL: PX600431; CHI: MK216764; CHS: ON462260.1), Lindsaea orbiculata (CHIL: MK216766; CHI: MK216765; CHS: MK216767), Solanum lycopersicum (CHIL: Solyc05g052240.3.1; CHI: Solyc05g010310.3.1; CHS: Solyc05g053550.3.1), Glycine max (CHIL: KRH53728; CHI: KRG92978; CHS: KRG94730), Oryza sativa (CHIL: Os11t0116300-01; CHI: Os03t0819600-01; CHS: Os11t0530600-01), Zea mays (CHIL: Zm00001eb163860_P001; CHI: Zm00001eb062510_P001; CHS: Zm00001eb322090_P001), Triticum aestivum (CHIL: TraesCS5B02G145800.1; CHI: TraesCS5A02G475600.1; CHS: TraesCS2B02G558400.1), Chlamydomonas reinhardtii (CHIL-like: XP_042928144.1; CHI-like: XP_042918416.1; CHS-like: XP_042914738.1), and Anabaena cylindrica (outgroup; CHIL-like: WP_015213863.1; CHI-like: WP_015214598.1; CHS-like: WP_015217128.1). Selected sequences were aligned using MAFFT49 for multiple sequence alignment. Phylogenetic trees were then generated using IQ-TREE250 with ultrafast bootstrap and SH-like approximate likelihood ratio test parameters (-bb 1000 -alrt 1000), with Anabaena cylindrica designated as the outgroup. Phylogenetic tree were visualized using the ggtree package51.

SKSA Algorithm Development and Implementation

To systematically identify and classify CHIL proteins across diverse plant species, we developed a Structure-based Key Amino acid residues clustering plus Sequence Alignment (SKSA) algorithm. This method integrates structural and sequence-based features to improve the specificity of CHIL protein prediction. The SKSA workflow operates through a two-step analytical process. First, it determines the sequence homology between a query protein and experimentally validated CHIL proteins using BLASTP and multiple sequence alignment. Second, for confirmed CHIL homologs, SKSA quantitatively evaluates their potential regulatory activity by analyzing the conservation patterns of key residues associated with CHI active sites and CHS-CHIL complex interfaces. This residue-level assessment provides a structural basis for predicting CHIL functional diversity and evolutionary conservation (Supplementary Fig. 11). The algorithm was implemented in Python, employing standard bioinformatics libraries for alignment and clustering. The full source code, datasets, and usage documentation are available on GitHub at https://github.com/xuzhougeng/SKSA. The code supporting this study has been deposited in Zenodo and is accessible via DOI: 10.5281/zenodo.18618601.

Single-nucleus RNA-seq data analysis

We reanalyzed single-nucleus RNA-seq (snRNA-seq) data from six developmental stages (S1-S6) of Arabidopsis thaliana leaves, sourced from a prior study30, where S3 represents the fully expanded mature stage and S4 marks the onset of senescence with marginal leaf yellowing (Supplementary Fig. 1). Epidermal (Epi) cell transcriptomes from S3 and S4 were integrated using Seurat v4.352 (R package). Following the standard Seurat workflow, the top 20 principal components were selected for dimensionality reduction and clustering, with cell clusters visualized via Uniform Manifold Approximation and Projection (UMAP). Differentially expressed genes (DEGs) between S3 and S4 were identified using the Wilcoxon test in FindMarkers52. The top 200 DEGs underwent KEGG pathway enrichment analysis using clusterProfiler53 (R package). For downstream analysis, we retained only cells with non-zero expression of both AT5G13930 and AT5G05270 genes and excluded cell types with fewer than three such cells. Hypergeometric tests (P < 0.01) on FPKM-normalized count data revealed significant enrichment of these genes in Epi cells at S1, S2, S3, and S6 (Supplementary Fig. 2). Pearson correlation analysis further revealed stage- and cell-type-specific co-expression patterns: significant positive correlations between AT5G13930 (CHS) and AT5G05270 (CHIL) were observed across all cell types at S1, S2, S3, and S6. Notably, mesophyll (Mes) cells showed significant correlations exclusively at S1 and S2, whereas epidermal (Epi) cells exhibited significance at S2, S3, and S6. By contrast, bundle sheath (BS) cells displayed significant correlation only at S3 (Supplementary Fig. 1).

Molecular dynamics (MD) simulations

To elucidate how CHIL affects different stages of the CHS catalytic process, we performed MD simulations on six systems corresponding to three representative binding states with or without CHIL: substrate-bound (p-coumaroyl-CoA or malonyl-CoA) and product-bound (NAR and CoA). Initial complex structures were prepared as follows. For CHS, we used the experimentally determined crystal structures available from this work (PDB ID: 9WBE [https://doi.org/10.2210/pdb9WBE/pdb]) to ensure accurate active site geometry. For the product-bound ligands NAR and CoA, binding poses were extracted from a previously resolved CHS-product complex (PDB ID: 7VF0 [https://doi.org/10.2210/pdb7VF0/pdb]) to maintain experimentally validated ligand positions. For other components (CHIL, p-coumaroyl-CoA, and malonyl-CoA), we used AlphaFold354 to predict the dimeric CHS-CHIL assemblies following the alignment to the CHS crystal structure for their coordinates. The predicted CHIL structure was used instead of the crystallographic model to avoid steric clashes with substrates or CoA observed in preliminary modeling. All systems were constructed using CHARMM-GUI (Version: 2.0)55,56 with the CHARMM36m57 force field for proteins and CGenFF4.658 for small molecules. Protonation states were assigned at pH 7.0 using PROPKA3.159. Systems were solvated with TIP3P water60 and neutralized with 0.15 M NaCl. The simulation boxes measured approximately 97 x 89 x 73 Å3 (without CHIL) or 121 x 100 x 91 Å3 (with CHIL), containing ~59,000 or ~104,000 atoms, respectively. Energy minimization was carried out using steepest-descent and adopted-basis Newton-Raphson algorithms61 (50 steps each). Solvent relaxation was performed under NVT conditions for 5 ps with restraint force constant of 400.0 kJ/mol/nm2 on protein backbone and small molecule heavy atoms and that of 40.0 kJ/mol/nm2 on protein sidechain atoms. Production simulations were conducted using OpenMM7.662 at 303.15 K and 1 bar with a 2 fs timestep. Long-range electrostatics were computed using the particle-mesh Ewald method63 with a 12 Å real-space cutoff, and Lennard-Jones interactions were smoothed between 10 Å and 12 Å. Hydrogen-involving bonds were constrained with SHAKE64. Each simulation was run for 200 ns and repeated three times with different random seeds. For each simulation, one frame was saved every 0.1 ns and 2000 frames were generated for analysis. Trajectory analyses, including RMSD and ligand movement quantification, were performed using MDAnalysis2.365. PyMOL3.1 was adopted to illustrate structures. In addition, to examine the potential role of CHIL in balancing the formation of naringenin chalcone versus derailment products, we constructed four additional CHS-CHIL systems bound with either the canonical products (NC and NAR) or the byproducts (BNY and CTAL). The CHS and CHIL structures were obtained from this study, while the bound small molecules were modeled using AlphaFold3 predictions (except NAR, taken from PDB ID: 7VF0 [https://doi.org/10.2210/pdb7VF0/pdb]). System preparation, force field parameters, and simulation protocols were identical to those described above. The data used in this study are publicly available in Zenodo at https://doi.org/10.5281/zenodo.18160349.

Statistics

Two-tailed Student’s t test and ordinary one-way ANOVA with uncorrected Fisher’s LSD test were performed to determine the statistical significance between different samples in quantification. Significant differences are indicated by different lettering at P<0.05 unless otherwise stated. All statistic results and graphs were generated by GraphPad Prism 10 (www.graphpad.com). The detailed descriptive statistics and results are given in Source Data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Jian-Xu Li and colleagues, describes the structural basis of the plant metabolic CHS-CHIL complex. By employing protein X-ray crystallography, the authors report the first example of the regulatory CHS-CHIL complex and reveal a detailed structural basis for CHS regulation by CHIL binding. This is a noteworthy result of high significance to the field, which has focused on the study of this complex in recent years. Protein pull-down experiments and enzyme assays combined with site-directed mutagenesis are used to validate the functional roles of the two distinct binding interfaces, apparent from the crystallographic structure. Evolutionary analysis by the authors reveals how functional, recombinant, purified CHS-CHIL complexes can be reconstituted from diverse plant lineages, including Marchantia. Through this work, the first structural evidence is presented describing how CHIL binding to CHS alters CHS product specificities, providing significant insight into a key molecular mechanism for the regulation of metabolic flux in the flavonoid pathway in plants.\nThe results described in the paper are interesting and could present a significant step toward understanding of the regulation of CHS by CHIL. The paper will be strengthened by addressing a number of significant concerns, listed below. There are currently issues around missing methodology, the need for proper controls for some experiments, including missing data, and adding appropriate experiments to adequately support conclusions.\n1. Figure 1a. CTAL is identified as a product, but no chemical standard is used, nor MS/MS data presented (in the main figures or SI), to support this claim. The main text states. “Through comparison with authentic standards and tandem MS/MS analysis, P1 and P2 were identified as the major product of (2S)-naringenin (an isomer of NC that forms via a non-enzymatic isomerization) and the byproduct CTAL, respectively.” Please include the missing MS/MS data to support the conclusion that CTAL is an identified here as a product. If no data is given to support the identification as CTAL, it should not be labeled as such. The need for validity of this characterization is critical, as also applies to results presented in figures 1e, 3e, 3i, 3g, 4f, 4g, 4h.\n2. Figure 1a. This experiment needs to include a standard for naringenin chalcone – the product of CHS. Naringenin chalcone has the same mass as naringenin and CTAL and must be clearly distinguished from the peaks shown by retention time.\n3. Throughout the paper, the protein pulldown method is used to validate protein-protein binding between recombinant CHS and CHIL (Figures 1c; 2d; 2f; 2h). A primary concern is that these pulldown results occur through non-specific binding, via nickel binding to CHS, instead of specific binding between CHIL-CHS. Please add experiments to ensure that non-specific binding is not repeatedly facilitating the observed pull-down result. For example, it is appropriate here to use the GST-CHS as bait for 6xHis-CHIL binding, since GST-glutathione is a more specific bimolecular interaction than 6xHis-nickel. To support conclusions of the CHS-CHIL binding interaction via pulldown, each experiment (Figures 1c; 2d; 2f; 2h) should show that the CHIL-CHS complex can be purified by GST pulldown. It should be clarified which proteins serve as bait in each experiment shown.\n4. For all gels presented in the main figures, the gels are cropped such that MW markers are not visible (Figures 1c; 2d; 2f; 2h; 4c). Please present the gels to include the MW markers to make them easier to interpret.\n5. In the current form, figures 3d, 3f do not provide convincing evidence of mutation-induced binding changes to support the following statement made in the main text: “As shown in Fig. 3h and 3i, the interactions between CHS and CHIL were severely impaired by H36A, F37A, and M4A (I35A/H36A/F37A/L38A).”\nIn figure 3d, and 3f, mutation-induced binding changes are not clear, as displayed solely from the CHS band intensity of Coomassie-stained gels. The bands would need to be quantified for comparison; however, there appears to be significant variability in CHIL and CHS loading of the gels (see CHIL input in 3f) that further confound the interpretation that the strength of the CHS band is reflective of relative strength of binding.\nThis pull-down method therefore appears insufficient to conclude how tested mutations affect CHS-CHIL binding interactions. To support conclusions that the tested mutations at ITF-1 and ITF-2 alter CHIL-CHS binding, quantitative binding experiments are needed (such as ITC).\n6. Two forms of CHS-CHIL complex data are presented in this paper: 1) solution CHS-CHIL data, and 2) Crystallographic CHS-CHIL data. It is not yet clear from experiments presented in the paper that the solution CHS-CHIL complex is indeed the same as the crystallographic CHS-CHIL complex. An additional SEC-MALS experiment is needed to validate the molecular weight of the native eluted complex in solution, in order to confirm that it matches the molecular weight of the heterotetrameric complex identified from the crystal structure. The SDS-PAGE gel (Figure 1d) is not sufficient evidence of a functional heterotetrameric assembly in solution.\n7. The SDS-PAGE gel (Figure 1d) is missing a loading lane.\n8. A CHIL dimer is labeled on the SDS-PAGE gel (Figure 1d), but a separate CHIL dimer peak is not evident from the chromatogram (Figure 1d). SEC-MALS is needed support the conclusion that the MW of this eluted fraction is consistent with a CHIL dimer.\n9. There is ambiguity in the methods section (In Vitro Pull-Down Assays and Western Blot) over which experiments use Coomassie staining vs. western blot. Please state in each figure caption whether Coomassie or western blot was used, for each gel shown.\n10. Gene sequence accession numbers are missing and need to be included for all sequences used for the phylogenetic tree analysis, as well as for the CHI enzyme used. Currently, no information is given about which CHI enzyme is used in Figure 1c and 1e. A gene accession number and statement of genus and species is needed for the CHI enzyme used.\n11. Information is missing about the sources of key materials: p-coumaroyl-CoA, malonyl-CoA, and naringenin chalcone.\n12. The paper is missing a description of the methodology and software used for the docking experiments (Figure 4a, 4b).\n13. Naringenin is shown in Figure 4e; it is unclear if it is docked here? Some description is needed to clarify this.\n14. The web-based analytical tool presented in the results section lacks sufficient information to review its significance, intended results or methodology. It should either be removed from this manuscript, or information added to comprehensively describe the tool and its outputs.", "reviewer_2": "Overall Evaluation\nWhile the elucidation of the crystal structure of the CHS-CHIL complex is a novel and interesting finding, the work's overall impact is significantly undermined by several critical flaws. The majority of the reported activity data is not new, and the authors' discussion of the mechanistic aspects of CHIL's action is based on flawed experimental design, misinterpretation of results, and logical inconsistencies.\nMajor Concerns\n1. The manuscript lacks essential quantitative and kinetic analyses of the enzymatic reaction. Such data are indispensable for a meaningful discussion of the results of mutational studies and the mechanistic role of CHIL in regulating CHS activity.\n2. The interpretation and logic concerning the mechanism of CHIL action are fatally flawed:\n· The authors observe the significant steric hinderance between the CHIL's beta-hairpin and coenzyme A (CoA) to propose that CHIL, which binds to the CHS active site (the CoA binding pocket), modulates product specificity by accelerating CoA release to increase reaction rates. However, the production of the secondary product (CTAL) also yields CoA molecules. Therefore, accelerating CoA release would also increase CTAL production, and cannot account for the observed specific activation of the primary product (NC (NAR)) production.\n· The starter and extender substrates for CHS also bind to the CoA binding pocket and would compete with CHIL. This competition should inhibit the CHS-catalyzed reaction, not activate it.\n· The authors' isolation of a stable CHS-CHIL complex strongly suggests a tight binding nature, likely with a dissociation constant in the nanomolar (nM) range or lower, consistent with the observation by other group. This implies that CHIL, after its binding to the CoA-binding pocket, would not readily dissociate from the complex and would therefore function as a tight-binding inhibitor, rather than an activator.\n· The proposed model of CHIL action, as described on lines 1-11 of page 12, is not supported by the experimental results and contains inconsistencies. For instance, the authors state that CHIL's binding \"opens the active pocket channel to permit substrate entry, subsequently promotes CoA release from CHS to enhance its catalytic activity, and meanwhile, the hydrophobic residues at the tip of CHIL’s beta-hairpin stabilize the reaction intermediates to increase the formation of the main product of CHS.\" This description raises a fundamental question: what experimental evidence supports these claims? The manuscript lacks data to substantiate that: (i) CHIL physically \"opens the active pocket channel to permit substrate entry.\" and (ii) Hydrophobic residues within CHIL's β-hairpin \"stabilize the reaction intermediates\" to specifically increase the formation of the main product. Without direct experimental evidence, these claims are speculative and undermine the core mechanistic argument of the paper.\n· Also, the manuscript lacks data to substantiate the description \"Notably, the beta-hairpin region of CHIL, which protrudes toward the entrance of CHS's substrate binding pocket, undergoes positional and conformational changes during CHS's catalytic reaction.\" There is no experimental evidence supports the motion of beta-hairpin region of CHIL during CHS catalysis.\n3. The findings described in the section titled \"Regulation of the activity and product specificity of CHS by CHIL\" are not new. They are either confirmatory or represent only incremental advances compared to previously reported findings, as seen in the cited references.\n4. Similarly, although the computational approaches and subsequent functional analyses presented in the section titled \"Evolution of CHIL\" were interesting, the findings obtained are essentially an incremental extension of previously published work and lack sufficient novelty.\n5. The manuscript suffers from a lack of experimental detail, which compromises the validity and reproducibility of the results:\n· The sources and purity of the chemicals used are not described. Without this critical information, the experimental results cannot be properly evaluated. For instance, some commercial p-coumaroyl-CoA preparations contain inhibitory impurities that can skew results. To mitigate this, standard CHS assays are often coupled with a 4-coumarate:CoA ligase-catalyzed reaction (see ref 12).\n· The gel filtration conditions used for the isolation of the CHS-CHIL complex need to be elaborated in detail.\n· Please provide CC1/2(%) values in Supplementary Table 1.", "reviewer_3": "The authors of this manuscript carried out a comprehensive mechanistic study addressing a long-standing question in flavonoid biosynthesis, particularly the function of CHIL and the product specificity of CHS (naringenin chalcone vs. the cyclization derailment product CTAL) when expressed alone in vitro. Using a combination of crystallography, biochemical assays, and evolutionary analyses, the authors demonstrate that CHIL proteins act as critical non-enzymatic partners that enhance the fidelity and efficiency of CHS, preventing the formation of derailment byproducts and promoting the specific synthesis of naringenin chalcone, the canonical precursor of flavonoids. The integration of in vitro enzyme kinetics, co-expression experiments, and mutant analyses confirms that CHIL fine-tunes CHS activity by stabilizing the active conformation and channeling intermediates toward the correct product. This work not only clarifies the elusive role of CHIL in flavonoid biosynthesis but also provides structural and mechanistic insights that explain discrepancies observed in earlier studies where CHS expressed alone yielded multiple non-physiological products. Overall, this is an important and well-written paper with well-executed experiments. I provide comments below with the intention of further strengthening and clarifying the paper, especially some of the key mechanistic claims.\n1. Page 10 “This biochemical evidence confirms an antagonistic relationship between CoA and CHIL, explaining our failure to obtain crystals of the CHS-CHIL complex with bound NAR and CoA despite several attempts. These findings suggested that CHIL modulates the activity and product specificity of CHS by accelerating CoA release to increase reaction rates.” This claim seems weak, as the biochemical observations presented earlier in the paragraph could support multiple mechanistic interpretations. It is intriguing that both CHS substrates, p-coumaroyl-CoA and malonyl-CoA, contain the large CoA moiety. If CHIL binding antagonizes CoA, how does initial p-coumaroyl-CoA binding, as well as the iterative malonyl-CoA binding, occur efficiently? Additionally, it is not specified whether the released CoA originates from p-coumaroyl-CoA or malonyl-CoA.\nI suggest the authors consider performing molecular dynamics (MD) simulations of CHS with and without CHIL, across different substrate, intermediate, or product-bound states. Such simulations could substantially enhance the mechanistic understanding of how CHIL modulates the complex catalytic sequence of this iterative enzyme. For example, they might reveal coordinated movements of the β-hairpin relative to the polyketide extension, cyclization, and product release steps.\n2. It remains unclear how the balance between the naringenin chalcone product and the CTAL derailment product is regulated in the presence or absence of CHIL. Insights into this could also potentially be gained through MD simulations.\n3. Did the authors observe smaller derailment products from single- or double-condensation events in addition to CTAL? If so, were these byproducts reduced in the presence of CHIL?\n4. Please ensure that ITF1 and ITF2 are properly defined upon first mention. What do these abbreviations stand for?"}, "author_response": "Point-by-point response to the referees’ comments\nDear Editor and Referees,\nWe appreciate your valuable comments on the manuscript (NCOMMS-25-\n61179A). All the comments and suggestions have been very carefully considered, and\nthe revised points are as listed below.\nReviewer #1 (Remarks to the Author):\nThe manuscript by Jian-Xu Li and colleagues, describes the structural basis of the plant\nmetabolic CHS-CHIL complex. By employing protein X-ray crystallography, the\nauthors report the first example of the regulatory CHS-CHIL complex and reveal a\ndetailed structural basis for CHS regulation by CHIL binding. This is a noteworthy\nresult of high significance to the field, which has focused on the study of this complex\nin recent years. Protein pull-down experiments and enzyme assays combined with site-\ndirected mutagenesis are used to validate the functional roles of the two distinct binding\ninterfaces, apparent from the crystallographic structure. Evolutionary analysis by the\nauthors reveals how functional, recombinant, purified CHS-CHIL complexes can be\nreconstituted from diverse plant lineages, including Marchantia. Through this work, the\nfirst structural evidence is presented describing how CHIL binding to CHS alters CHS\nproduct specificities, providing significant insight into a key molecular mechanism for\nthe regulation of metabolic flux in the flavonoid pathway in plants.\nThe results described in the paper are interesting and could present a significant\nstep toward understanding of the regulation of CHS by CHIL. The paper will be\nstrengthened by addressing a number of significant concerns, listed below. There are\ncurrently issues around missing methodology, the need for proper controls for some\nexperiments, including missing data, and adding appropriate experiments to adequately\nsupport conclusions.\nReply: We sincerely appreciate the reviewer’s thoughtful comments and positive\n1 / 39\nevaluation of our study. Your insights have greatly contributed to improving the clarity\nand rigor of our manuscript. Below, we address each of the points raised in detail.\n1. Figure 1a. CTAL is identified as a product, but no chemical standard is used, nor\nMS/MS data presented (in the main figures or SI), to support this claim. The main text\nstates. “Through comparison with authentic standards and tandem MS/MS analysis, P1\nand P2 were identified as the major product of (2S)-naringenin (an isomer of NC that\nforms via a non-enzymatic isomerization) and the byproduct CTAL, respectively.”\nPlease include the missing MS/MS data to support the conclusion that CTAL is an\nidentified here as a product. If no data is given to support the identification as CTAL, it\nshould not be labeled as such. The need for validity of this characterization is critical,\nas also applies to results presented in figures 1e, 3e, 3i, 3g, 4f, 4g, 4h.\nReply: We appreciate this important point and agree that further clarification was\nnecessary. The mass spectra of (2S)-Naringenin and CTAL were supplemented the\nrevised manuscript with Extended Data Fig. 4 (as shown below). Although we don't\nhave the chemical standard of CTAL, it’s mass spectra almost perfect match the\nreported mass spectra of CTAL (Supplementary Fig. 7 in Waki et al., Nat Commun,\n2020).\nExtended Data Fig. 4 | The mass spectra of peaks 1 and 2 (P1 and P2) in Fig.1b\nwere analyzed by tandem MS/MS spectrometry.\na, Mass spectra of the major product (2S)-Naringenin (NAR). b, Mass spectra of the\nbyproduct CTAL.\n2 / 39\nFor ease of reference, the figure from the cited article is reproduced below\n(Supplementary Fig. 7 in Waki et al., Nat Commun, 2020).\nSupplementary Fig. 7. Analyses of the products of the AmCHS-catalyzed reaction\nwith p-coumaroyl-CoA and malonyl-CoA in the presence and absence of AmCHIL. (a)\nHPLC chromatograms of the products of the AmCHS-catalyzed reaction with p-\ncoumaroyl-CoA and malonyl-CoA in the presence (lower panel) and absence (upper\npanel) of AmCHIL. Chromatograms were monitored by mass spectrometry at m/z of\n271 (blue line) and 229 (orange line). The THC produced via the CHS-catalyzed\nreaction was non-enzymatically and fully converted to naringenin under the\nexperimental conditions. (b) The MS spectra of peaks 1 and 2 (P1 and P2) in panel (a)\nwere analyzed by tandem MS spectrometry (left) and compared with those of CTAL\nprepared via the HmCTAS-catalyzed reaction with p-coumaroyl-CoA and malonyl-\nCoA (upper right) and naringenin (lower right).\nReferences:\nWaki, T. et al. A conserved strategy of chalcone isomerase-like protein to rectify\npromiscuous chalcone synthase specificity. Nat Commun 11, 870 (2020).\n2. Figure 1a. This experiment needs to include a standard for naringenin chalcone -\nthe product of CHS. Naringenin chalcone has the same mass as naringenin and CTAL and\n3 / 39\nmust be clearly distinguished from the peaks shown by retention time.\nReply: We thank the reviewer for the suggestion. In the enzyme activity assay of\nchalcone synthase (CHS), the primary analyte of interest is typically naringenin, an\nisomer derived from naringenin chalcone (NC), through non-enzymatic isomerization.\nThis preference arises because, in the absence of the enzyme, naringenin chalcone\nundergoes spontaneous, non-stereospecific C-ring closure to yield naringenin (Abe et\nal., J Am Chem Soc, 2000). It is common in the literature for CHS activity to be\nevaluated based on the detection of naringenin (Waki et al., Nat Commun, 2020; Niu et\nal., Front Plant Sci, 2021; Ni et al., J Integr Plant Biol, 2022).\nTo verify your suggestion that naringenin chalcone may also be present in the CHS\nenzymatic reaction products, we developed an HPLC-MS method capable of resolving\nnaringenin chalcone from naringenin. The experimental results are presented in the\nFig.R1 below. Our analysis confirmed the absence of naringenin chalcone following\nthe enzymatic reaction, a finding that aligns with previous literature reports.\nFig. R1 | Extracted ion chromatogram (EIC) of in vitro enzymatic assays products\nwith proteins of the recombinant CHS. P1, (2S)-Naringenin; P2, Naringenin\nChalcone; NAR-STD, (2S)-Naringenin authentic standard; NC-STD, Naringenin\nChalcone authentic standard.\n4 / 39\nReferences:\n[1]. Waki, T. et al. A conserved strategy of chalcone isomerase-like protein to rectify\npromiscuous chalcone synthase specificity. Nat Commun 11, 870 (2020).\n[2]. Abe, I., Morita, H., Nomura, A. & Noguchi, H. Substrate specificity of chalcone\nsynthase: Enzymatic formation of unnatural polyketides from synthetic cinnamoyl-\nCoA analogues. J Am Chem Soc 122, 11242-11243 (2000).\n[3]. Ni, R. et al. Molecular and structural characterization of a promiscuous chalcone\nsynthase from the fern species Stenoloma chusanum. J Integr Plant Biol 64, 1935-\n1951 (2022).\n[4]. Niu, M. et al. Functional and Structural Investigation of Chalcone Synthases Based\non Integrated Metabolomics and Transcriptome Analysis on Flavonoids and\nAnthocyanins Biosynthesis of the Fern Cyclosorus parasiticus. Front Plant Sci 12,\n757516 (2021).\n3. Throughout the paper, the protein pulldown method is used to validate protein-\nprotein binding between recombinant CHS and CHIL (Figures 1c; 2d; 2f; 2h). A\nprimary concern is that these pulldown results occur through non-specific binding,\nvia nickel binding to CHS, instead of specific binding between CHIL-CHS. Please add\nexperiments to ensure that non-specific binding is not repeatedly facilitating the\nobserved pull-down result. For example, it is appropriate here to use the GST-CHS as\nbait for 6xHis-CHIL binding, since GST-glutathione is a more specific bimolecular\ninteraction than 6xHis-nickel. To support conclusions of the CHS-CHIL binding\ninteraction via pulldown, each experiment (Figures 1c; 2d; 2f; 2h) should show that the\nCHIL-CHS complex can be purified by GST pulldown. It should be clarified which\nproteins serve as bait in each experiment shown.\nReply: We thank the reviewer for pointing out this important methodological\nconsideration. To determine whether non-specific binding under our experimental\nconditions could affect the protein pull-down results, we expressed the GST-tagged\nproteins, including the GST-tag alone, CHS wild-type, and its mutant forms, and\n5 / 39\nperformed purification using both GST-resin (Glutathione Sepharose) and Nickel-resin\n(Ni-NTA Agarose) (see Fig.R2 below). The results showed that the GST-resin\neffectively enriched the target GST-tagged proteins. As the reviewer noted, some non-\nspecific binding was observed with the Nickel-resin; however, the non-specific bands\nwere minimal and did not compromise the interpretation of the pull-down experiments\nof CHS and CHIL in our manuscript.\nFig. R2 | SDS-PAGE analysis to illustrate the binding effects of GST-tag alone,\nCHS wild-type, and its mutant forms with GST-resin (Glutathione Sepharose) and\nNickel-resin (Ni-NTA Agarose).\nTo address the reviewer's comments, we experimentally evaluated whether the\nCHIL-CHS complex can be purified using GST pull-down (see Fig.R3 below). The\nexperimental results indicate that the CHIL-CHS complex can be purified by GST\npulldown, but CHI does not bind to CHS.\nFig.R3 | SDS-PAGE analysis of the CHIL-CHS complex purified by GST pull-\n6 / 39\ndown.\nAdditionally, for the pull-down experiments in Fig. 1c, Fig. 3 (d, h, f), and Fig. 4c,\na 6x His-tagged CHIL protein was used as the bait. This information is now explicitly\nprovided in the corresponding figure legends. We are grateful for your kind advice.\n4. For all gels presented in the main figures, the gels are cropped such that MW markers\nare not visible (Figures 1c; 2d; 2f; 2h; 4c). Please present the gels to include the MW\nmarkers to make them easier to interpret.\nReply: Thank you very much for your suggestions. When cropping the images in Fig.1c\nand Fig.4c, we retained the molecular weight markers, as indicated in the revised Fig.1\nand Fig.4. The updated versions of Fig.1c and Fig.4c are provided below. For Fig.2d,\n2f, and 2h, we have included supplementary figure (Extended Data Fig. 6) to provide\na more detailed explanation, as shown in the attached images below.\nFig.1c, SDS-PAGE illustrating the binding ability between CHS with CHI and CHIL\ndetermined through pull-down experiments. CHS was fused to glutathione S-\ntransferase (GST-tag), and CHIL and CHI were fused to 6xHis-tag.\n7 / 39\nFig.4c, CoA affects the interaction between CHS and CHIL.\nExtended Data Fig. 6 | Quantitative analysis of the protein bands in the pull-down\nassay shown in Fig. 3.\na, Quantitative analysis of the data in Fig.3d. b, Quantitative analysis of the data in\nFig.3f. c, Quantitative analysis of the data in Fig.3h.\n5. In the current form, figures 3d, 3f do not provide convincing evidence of mutation-\ninduced binding changes to support the following statement made in the main text: “As\nshown in Fig. 3h and 3i, the interactions between CHS and CHIL were severely\nimpaired by H36A, F37A, and M4A (I35A/H36A/F37A/L38A).”\nReply: Thank you for pointing this out. As the quantitative analysis of protein bands\nindicated that the H36A mutation attenuated the interaction between CHS and CHIL\n(Extended Data Fig. 6c.), we have accordingly revised the corresponding description\nin the manuscript.\nFurthermore, following your suggestion below, we also performed ITC assays to\nquantify the interaction between CHS and CHIL (Extended Data Fig. 8.). The figure\nis shown below. The results confirmed that the H36A mutant of CHIL still binds to\n8 / 39\nCHS. However, the binding affinity was weakened, as indicated by a 48% increase in\nthe K value.\nD\nIn figure 3d, and 3f, mutation-induced binding changes are not clear, as displayed solely\nfrom the CHS band intensity of Coomassie-stained gels. The bands would need to be\nquantified for comparison; however, there appears to be significant variability in CHIL\nand CHS loading of the gels (see CHIL input in 3f) that further confound the\ninterpretation that the strength of the CHS band is reflective of relative strength of\nbinding.\nReply: We appreciate this important point and agree that further quantitative\nanalysis was necessary. Accordingly, in response to the comments, we have provided a\nquantitative analysis of the protein bands in the pull-down assay (Fig. 3d, 3f, 3h),\nas shown in Extended Data Fig. 6. The results indicated that several mutations impaired\nthe CHS-CHIL interaction. In particular, the K97A, Q100A, Q104A, Y138A, and M3A\n(K94A/K97A/Q100A) mutations caused relatively severe impairment, while the K94A\nmutation only led to a slight reduction.\nExtended Data Fig. 6 | Quantitative analysis of the protein bands in the pull-down\nassay shown in Fig. 3.\na, Quantitative analysis of the data in Fig.3d. b, Quantitative analysis of the data in\nFig.3f. c, Quantitative analysis of the data in Fig.3h.\nThis pull-down method therefore appears insufficient to conclude how tested mutations\naffect CHS-CHIL binding interactions. To support conclusions that the tested mutations\nat ITF-1 and ITF-2 alter CHIL-CHS binding, quantitative binding experiments are\nneeded (such as ITC).\n9 / 39\nReply: We thank the reviewer for this valuable suggestion. As suggested by the\nreviewers, we performed ITC experiments on several mutants of the CHIL protein from\nITF-1 and ITF-2 (Extended Data Fig. 8). The results showed that the binding affinity\nbetween CHIL and CHS was 7.35 μM. Compared to wild-type CHIL, the H36A mutant\nstill bound to CHS, but with weakened binding affinity, as reflected by a 48% increase\nin the K value. In contrast, no detectable binding was observed for the F37A, M4A,\nD\nK97A, Q100A, or M3A mutants of CHIL, as no reliable K values could be determined.\nD\nNotably, the H36L mutant, which significantly enhances the regulatory capacity of\nCHIL toward CHS, exhibited an approximately 3.6-fold increase in binding affinity.\nAccordingly, we have revised the relevant paragraphs in the manuscript.\nExtended Data Fig. 8 | ITC measurement of CHS binding affinity to CHIL and its\nmutants. Titration curves for CHIL (wild-type) and the mutants (H36L, H36A, F37A,\nM4A, K97A, Q100A, M3A) are colored in red, purple, blue, green, cyan, brown, yellow,\nand gray, respectively.\n10 / 39\n6. Two forms of CHS-CHIL complex data are presented in this paper: 1) solution CHS-\nCHIL data, and 2) Crystallographic CHS-CHIL data. It is not yet clear from\nexperiments presented in the paper that the solution CHS-CHIL complex is indeed\nthe same as the crystallographic CHS-CHIL complex. An additional SEC-MALS\nexperiment is needed to validate the molecular weight of the native eluted complex in\nsolution, in order to confirm that it matches the molecular weight of the\nheterotetrameric complex identified from the crystal structure. The SDS-PAGE gel\n(Figure 1d) is not sufficient evidence of a functional heterotetrameric assembly in\nsolution.\nReply: Thanks to the constructive suggestions from the reviewer. Accordingly, we\nhave performed two complementary experiments: (1) Size exclusion chromatography\n(SEC) analysis of CHS and the CHS-CHIL complex (Fig.R4 below), and (2) Cryo-EM\nanalysis of the complex. (Fig.R5 below). This approach was taken because the CHS-\nCHIL complex lacks the stability required for long-distance shipping to an alternative\nSEC-MALS facility, whose access was further limited by maintenance of our routine\nused SEC-MALS instrument.\nWe first purified GST-tagged CHS and its complex with His-tagged CHIL. Size\nexclusion chromatography (SEC) analysis showed that the co-purified sample eluted\napproximately 0.7 mL earlier than CHS alone, indicating the formation of a stable CHS-\nCHIL complex in solution (Fig.R4 below). To verify whether this solution-state\ncomplex corresponds structurally to the crystallographic CHS-CHIL complex, we\nproceeded with cryo-EM sample preparation and collected data using the 300kV cryo-\nelectron microscope. Although the dataset was limited in size, we performed single-\nparticle 2D classification, which unambiguously confirmed that the solution-state CHS-\nCHIL complex adopts the same heterotetrameric architecture as observed in the crystal\nstructure (Fig.R5 below).\n11 / 39\nFig. R4 | SEC profiles of GST-tagged CHS and its complex with His-tagged CHIL.\nFig. R5 | 2D classification of CHS-CHIL complex.\n7. The SDS-PAGE gel (Figure 1d) is missing a loading lane.\nReply: We thank the reviewer for pointing this out. As suggested, we have added a\nloading lane to the SDS-PAGE gel in Fig.1d, as shown below.\nFig.1d, Size-exclusion chromatogram and Coomassie-blue stained SDS-PAGE of\nCHS-CHIL complex.\n8. A CHIL dimer is labeled on the SDS-PAGE gel (Figure 1d), but a separate CHIL\ndimer peak is not evident from the chromatogram (Figure 1d). SEC-MALS is needed\n12 / 39\nsupport the conclusion that the MW of this eluted fraction is consistent with a CHIL\ndimer.\nReply: We are grateful for the reviewer's insightful comments and valuable suggestions.\nTo confirm the identity of the band marked as the CHIL dimer, we subjected it to\nprotein Mass Spectrometry. The results confirmed that the protein is indeed CHIL\n(Q8VZW3), as shown in the first row of Fig.R6 below.\nFig. R6 | Mass spectrometric identification of the band corresponding to the CHIL\ndimer.\nTo explain the presence of the CHIL dimer in gels, we hypothesized oxidative\ndimerization of monomers. We observed that the dimer band is faint in fresh samples\nbut intensified upon sample aging. This hypothesis was verified by protein Mass\nSpectrometry, which identified an intermolecular disulfide bond (Fig. R7 below). Thus,\nwe conclude that fresh samples are imperative for the correct characterization of CHIL\nand its complex with CHS.\nFig. R7 | Disulfide Bond in CHIL Dimer Identified by Mass Spectrometry.\n13 / 39\n9. There is ambiguity in the methods section (In Vitro Pull-Down Assays and Western\nBlot) over which experiments use Coomassie staining vs. western blot. Please state in\neach figure caption whether Coomassie or western blot was used, for each gel shown.\nReply: We thank the reviewer for pointing this out. We apologize for the oversight in\nour description. To confirm the specificity of the target protein bands in the pull-down\nassays, we did perform a complementary Western Blot analysis. The results verified\nthat the bands were indeed the correct targets. However, as the final data presented in\nthe manuscript are based on Coomassie staining, we have now revised the relevant text\nto accurately reflect this and avoid any confusion.\n10. Gene sequence accession numbers are missing and need to be included for all\nsequences used for the phylogenetic tree analysis, as well as for the CHI enzyme used.\nCurrently, no information is given about which CHI enzyme is used in Figure 1c and\n1e. A gene accession number and statement of genus and species is needed for the CHI\nenzyme used.\nReply: We appreciate the reviewer's valuable comment. The CHI enzyme analyzed in\nFigures 1c and 1e corresponds to the gene accession number AT3G55120.1. In response,\nwe have now incorporated the relevant gene accession numbers into the respective\nfigure legends (including Fig. 1, Fig. 5a, and the Supplementary Figures). All\nmodifications have been implemented in the revised version of the manuscript.\n11. Information is missing about the sources of key materials: p-coumaroyl-CoA,\nmalonyl-CoA, and naringenin chalcone.\nReply: We acknowledge the importance of this point raised by the reviewer. The key\nreagents p-coumaroyl-CoA (Catalog No. S25276), naringenin (Catalog No. B21596),\nand naringenin chalcone (Catalog No. B21598) were purchased from Shanghai YuanYe\nBio-Technology Co., Ltd. Malonyl-CoA (Catalog No. M4263) was obtained from\nSigma-Aldrich. As suggested, we have incorporated this information into the Methods\nsection of our revised manuscript.\n14 / 39\n12. The paper is missing a description of the methodology and software used for the\ndocking experiments (Figure 4a, 4b).\nReply: We are grateful to the reviewer for their insightful comment. We have now\nprovided the relevant methodological description in the revised manuscript. The\ndetailed analytical methods are described in our response to Question 13, as provided\nbelow.\n13. Naringenin is shown in Figure 4e; it is unclear if it is docked here? Some description\nis needed to clarify this.\nReply: We sincerely thank the reviewer for this insightful suggestion. We\nsuperimposed our CHIL-CHS complex structure with the previously reported CHS-\nCoA-naringenin complex structure (CpCHS, PDB: 7VF0) in Figure. 4a, 4b, and 4e. The\nanalysis revealed that the overall structure of CHS in the CHIL-CHS complex closely\nresembles those of AtCHS and CpCHS, including the naringenin- and CoA-binding\npockets. Therefore, we modeled the CoA and/or naringenin molecules from 7VF0 into\nthe CHIL-CHS complex structure to facilitate further analysis. The corresponding\nmethod description has been added to the revised manuscript.\n14. The web-based analytical tool presented in the results section lacks sufficient\ninformation to review its significance, intended results or methodology. It should either\nbe removed from this manuscript, or information added to comprehensively describe\nthe tool and its outputs.\nReply: We agree with the reviewer. The web-based analytical tool is provided solely\nto facilitate community use of our SKSA procedure and was not used to generate\nprimary results. Accordingly, we have removed its description from the Results and\nrelocated it to the Methods section (\"SKSA Algorithm Development and\nImplementation\"), where we now provide detailed information on the algorithmic steps\nand implementation. The complete source code has also been released on GitHub\n(https://github.com/xuzhougeng/SKSA) to ensure transparency and reproducibility.\n15 / 39\nWe sincerely thank the reviewer for their thorough and insightful comments. Your\nconstructive feedback has greatly helped us improve the clarity and rigor of our\nmanuscript. We have carefully addressed all the concerns raised and believe the\nrevisions have strengthened our work. We deeply appreciate the time and effort you\nhave dedicated to evaluating our study and the valuable suggestions you have provided.\n16 / 39\nReviewer #2 (Remarks to the Author):\nOverall Evaluation\nWhile the elucidation of the crystal structure of the CHS-CHIL complex is a novel and\ninteresting finding, the work's overall impact is significantly undermined by several\ncritical flaws. The majority of the reported activity data is not new, and the authors'\ndiscussion of the mechanistic aspects of CHIL's action is based on flawed experimental\ndesign, misinterpretation of results, and logical inconsistencies.\nReply: We thank the reviewer for their thoughtful assessment of our manuscript and\nfor acknowledging the novelty of the CHS-CHIL complex structure. We also appreciate\nthe constructive critiques regarding the activity data and mechanistic interpretation.\nThese comments have provided a valuable opportunity to conduct additional\nexperiments and substantially strengthen the study, as detailed below.\nWe agree with the reviewer that part of the kinetic parameters analysis served to\nestablish the functional context for the structural findings. In response, we have now\nperformed further kinetic analyses to more rigorously validate our conclusions. The\nnew results, included in Supplementary Table 2 and the main text, consistently support\nour initial findings and confirm the robustness of the observed trends. The manuscript\nhas been revised to clearly distinguish between the previously established data and the\nnew validation experiments.\nFurthermore, we have carried out extensive molecular dynamics (MD) simulations\nto independently probe the dynamic behavior of the complex. These results, now\npresented as Extended Data Fig. 14, 15 and 16, provide novel insights into the\ninteraction dynamics and functionally bolster the structural observations.\nOnce again, we are grateful for the reviewer’s rigorous and insightful feedback.\nThe additional kinetic analyses and MD simulations undertaken in response have\ngreatly deepened the mechanistic understanding and enhanced the overall clarity and\nimpact of the manuscript. We hope that the revisions and explanations provided here\n17 / 39\nfully address the reviewer’s concerns.\nMajor Concerns\n1. The manuscript lacks essential quantitative and kinetic analyses of the enzymatic\nreaction. Such data are indispensable for a meaningful discussion of the results of\nmutational studies and the mechanistic role of CHIL in regulating CHS activity.\nReply:\nWe appreciate this important point and agree that further clarification was necessary.\nFor malonyl-CoA, the enzymatic kinetic parameters were characterized for individual\nCHS, as well as for CHS in combination with wild-type CHIL and representative CHIL\nmutants (see Supplementary Table 2 below). The results demonstrate that CHIL\naddition markedly enhanced CHS catalytic efficiency, increasing both V and k by\nmax cat\napproximately 88%, while also raising the K value by about 74%. Kinetic\nm\ncharacterization of CHIL mutants further revealed distinct functional effects: the H36L\nmutant, which enhances CHS activation compared to wild-type CHIL, elevated V\nmax\nand k by an additional 38%, indicating a strengthened regulatory capacity. In contrast,\ncat\ntwo binding-deficient mutants, M4A located at ITF1 and Q100A located at ITF2,\nexhibited kinetic parameters similar to those measured in the absence of CHIL. This\nsupports the conclusion that CHIL-CHS interaction is essential for the observed\ncatalytic enhancement. Similarly, for p-coumaroyl-CoA, CHIL addition significantly\nimproved CHS catalytic efficiency, with V and k increasing by approximately 56%\nmax cat\nand K rising by about 77%. The H36L mutant further enhanced this effect, elevating\nm\nV\nmax\nand k\ncat\nby an additional 69% relative to wild-type CHIL.\nOur kinetic analysis reveals that CHIL increases the K of CHS for both substrates\nm\nwhile maintaining comparable k /K ratios. We emphasize that k /K primarily\ncat m cat m\nreflects catalytic efficiency near zero substrate concentration, which differs\nsubstantially from actual metabolic conditions. In enzyme-substrate condensates like\nmetabolons, substrate concentrations remain high relative to K . Under such conditions,\nm\n18 / 39\nk becomes the primary determinant of catalytic rate. Consequently, CHIL's\ncat\nenhancement of k provides a plausible mechanism for its function in accelerating\ncat\nproduct synthesis in physiological environments. Similar results have been reported in\nprevious studies (Waki et al., Nat Commun, 2020).\nSupplementary Table 2. Kinetic parameters of CHS-catalyzed NAR production\nwith malonyl-CoAa and p-coumaroyl-CoAb in the absence and presence of CHIL\nvariants. a50 M p-coumaroyl-CoA was used as the starter substrate,b100 M malonyl-\nCoA was used as the extender substrate.\nReferences:\nWaki, T. et al. A conserved strategy of chalcone isomerase-like protein to rectify\npromiscuous chalcone synthase specificity. Nat Commun 11, 870 (2020).\n2. The interpretation and logic concerning the mechanism of CHIL action are fatally\nflawed:\n· The authors observe the significant steric hinderance between the CHIL's beta-hairpin\nand coenzyme A (CoA) to propose that CHIL, which binds to the CHS active site (the\nCoA binding pocket), modulates product specificity by accelerating CoA release to\nincrease reaction rates. However, the production of the secondary product (CTAL) also\nyields CoA molecules. Therefore, accelerating CoA release would also increase CTAL\nproduction, and cannot account for the observed specific activation of the primary\nproduct (NC (NAR)) production.\n19 / 39\nReply: We fully agree with the reviewer's insightful observation that accelerated CoA\nrelease alone would be expected to generally promote the formation of both major\nproduct and byproduct. In our model, steric-hindrance-mediated CoA release acts\nprimarily as a general rate-enhancement mechanism, increasing overall catalytic\nturnover by alleviating product inhibition. The key factor underlying the observed\nproduct specificity, however, is CHIL's distinct capacity to selectively stabilize the\npolyketide intermediate in a conformation that favors cyclization toward the major\nproduct (NC/NAR). The key to resolving the specificity paradox lies in the distinct\ninteraction patterns of CHIL's -hairpin. Our simulations reveal that while this hairpin\nis highly dynamic (rotating up to 60°), it selectively interacts with CoA (forming\nhydrogen bonds with E34 and F37 with occupancies of 47.0% and 5.9%, respectively)\nbut has minimal interaction with the substrate moieties of p-coumaroyl-CoA or\nmalonyl-CoA (Extended Data Fig. 14, 15 and 16). This specific interaction profile,\ncombined with the hairpin's flexibility, provides a mechanistic basis for our model: the\n-hairpin is structurally poised to discriminate between and stabilize specific polyketide\nintermediates. As supported by MD simulations and our experimental data, this specific\nstabilization actively redirects the reaction trajectory away from the derailment pathway\nleading to CTAL. Therefore, while CoA release enhancement boosts total productivity,\nthe selective stabilization of the major product's catalytic pathway is what drives the\nshift in product specificity observed in our experiments.\n· The starter and extender substrates for CHS also bind to the CoA binding pocket and\nwould compete with CHIL. This competition should inhibit the CHS-catalyzed reaction,\nnot activate it.\nReply: We thank the reviewer for raising this critical point regarding the steric\ncompetition between the CHIL and the CoA moiety of the substrates. Our structural\ndata indicate that these molecules compete for overlapping spatial regions-an\ninteraction that would conventionally imply inhibition. However, we propose that this\napparent competition is in fact integral to CHIL's activation mechanism: although CHIL\n20 / 39\nbinding initially displaces CoA, its subsequent engagement remodels the active site into\na superior catalytic state. In this reconfigured environment, the polyketide intermediate\nis stabilized to favor cyclization into the primary product, while CoA release is\nmarkedly accelerated. The net activation observed in kinetic assays thus reflects a trade-\noff in which the catalytic advantages conferred by the CHIL-bound state substantially\noutweigh the initial competitive binding penalty-effectively repurposing a typical\ninhibitory interaction into a refined activation switch.\n· The authors' isolation of a stable CHS-CHIL complex strongly suggests a tight binding\nnature, likely with a dissociation constant in the nanomolar (nM) range or lower,\nconsistent with the observation by other group. This implies that CHIL, after its binding\nto the CoA-binding pocket, would not readily dissociate from the complex and would\ntherefore function as a tight-binding inhibitor, rather than an activator.\nReply: We appreciate the reviewer's thoughtful perspective on the possible inhibitory\nrole of CHIL. Our experimental observations, while differing from this expectation,\nmay offer additional insights into the mechanism. The binding affinity measured by\nIsothermal Titration Calorimetry (ITC, K = 7.35 M) suggests a moderate interaction\nD\nstrength rather than tight binding (see Extended Data Fig. 8 below). This is\ncomplemented by our finding that CoA can partially displace CHIL from the complex\n(Fig.4c), indicating a reversible binding mode. Most importantly, the enzymatic kinetic\nparameters measurements consistently show enhanced catalytic efficiency of CHS\nupon CHIL binding. While the complete mechanistic picture continues to emerge, these\ncollective results lead us to propose that CHIL functions as a regulatory activator\nthrough a reversible interaction, rather than as a static inhibitor.\n21 / 39\nExtended Data Fig. 8 | ITC measurement of CHS binding affinity to CHIL and its\nmutants. Titration curves for CHIL (wild-type) and the mutants (H36L, H36A, F37A,\nM4A, K97A, Q100A, M3A) are colored in red, purple, blue, green, cyan, brown, yellow,\nand gray, respectively.\n· The proposed model of CHIL action, as described on lines 1-11 of page 12, is not\nsupported by the experimental results and contains inconsistencies. For instance, the\nauthors state that CHIL's binding \"opens the active pocket channel to permit substrate\nentry, subsequently promotes CoA release from CHS to enhance its catalytic activity,\nand meanwhile, the hydrophobic residues at the tip of CHIL’s beta-hairpin stabilize the\nreaction intermediates to increase the formation of the main product of CHS.\" This\ndescription raises a fundamental question: what experimental evidence supports these\nclaims? The manuscript lacks data to substantiate that: (i) CHIL physically \"opens the\nactive pocket channel to permit substrate entry.\" and (ii) Hydrophobic residues within\nCHIL's β-hairpin \"stabilize the reaction intermediates\" to specifically increase the\nformation of the main product. Without direct experimental evidence, these claims are\nspeculative and undermine the core mechanistic argument of the paper\nReply: We thank the reviewer for this important comment regarding the mechanistic\ninterpretation of CHIL's function. In response, we have moved the detailed mechanistic\n22 / 39\nproposal from the Results to the Discussion section to better frame it as a testable model\nconsistent with our experimental findings, rather than as an established conclusion. This\nadjustment ensures our presentation remains appropriately cautious while still allowing\nus to present a coherent mechanistic hypothesis for future investigation.\nThe proposed model is supported by several converging lines of evidence from\nour study: First, structural comparisons reveal conformational changes in CHIL's -\nhairpin between its free and CHS-bound states. Second, alanine mutations of key -\nhairpin residues (including H36 and F37) significantly reduce both binding affinity (as\nquantified by ITC and pull-down assays) and CHS activation. Third, MD simulations\nconsistently indicate the high flexibility of the -hairpin (Extended Data Fig. 14, 15 and\n16) and substrate stabilization in the presence of CHIL. Finally, the dramatic increase\nin k and V upon CHIL binding functionally demonstrates enhanced catalytic\ncat max\nefficiency.\nWhile we acknowledge that direct structural evidence for active-site opening and\nintermediate stabilization requires further investigation, the consistent correlation\nbetween structural data, binding affinity measurements, computational simulations, and\nfunctional outcomes provides a reasonable foundation for our proposed mechanism.\nWe believe this model, now appropriately positioned in the Discussion, offers a\nvaluable framework for guiding future research into CHIL's allosteric regulation of\nCHS.\n· Also, the manuscript lacks data to substantiate the description \"Notably, the beta-\nhairpin region of CHIL, which protrudes toward the entrance of CHS's substrate\nbinding pocket, undergoes positional and conformational changes during CHS's\ncatalytic reaction.\" There is no experimental evidence supports the motion of beta-\nhairpin region of CHIL during CHS catalysis.\nReply: We are grateful for the reviewer's insightful comment, which helps us refine the\npresentation of our structural findings. The description of -hairpin dynamics was\n23 / 39\nproposed as a reasonable interpretation derived from conformational differences\nobserved between the crystal structures of the CHS-CHIL complex and isolated CHIL.\nWhile our current experimental data do not directly demonstrate real-time movement\nof the -hairpin during catalysis, the structural comparison provides a plausible basis\nfor hypothesizing its functional relevance. In the revised manuscript, we clearly states\nthat the conformational difference between the two static structures suggests the -\nhairpin's capacity for movement, and that this flexibility is compatible with or may\nfacilitate the proposed model, rather than asserting its motion as a directly proven event\nduring catalysis. We believe this revision aligns our claims more precisely with the\navailable experimental data.\n3. The findings described in the section titled \"Regulation of the activity and product\nspecificity of CHS by CHIL\" are not new. They are either confirmatory or represent\nonly incremental advances compared to previously reported findings, as seen in the\ncited references.\nReply: We appreciate the reviewer's comment. We agree that previous studies have\nsuggested a functional link between CHS and CHIL. However, the objective of this\nsection was not merely to repeat these observations, but to establish a comprehensive\nand quantitative experimental foundation that directly enables the structural and\nengineering insights presented later in the manuscript. To this end, we systematically\nprovided several key pieces of new evidence that were previously unavailable: 1) We\nidentified a stage- and cell-type-specific co-expression pattern between CHS and CHIL,\noffering physiological context for their interaction; 2) We unambiguously demonstrated\nthe specificity of the physical interaction by confirming CHS-CHIL binding via pull-\ndown and gel filtration, while showing that CHS does not interact with CHI under the\nsame conditions; 3) We quantitatively verified that this specific CHIL binding enhances\nboth CHS catalytic activity and product specificity. This systematic analysis helps lay\nthe groundwork for and understand the mechanism behind the subsequent structural\nfindings. Our integrated analysis thus connects specific biochemistry to cellular context\n24 / 39\nand ultimately to molecular mechanism, forming a cohesive narrative from\nphysiological correlation to structural explanation.\n4. Similarly, although the computational approaches and subsequent functional\nanalyses presented in the section titled \"Evolution of CHIL\" were interesting, the\nfindings obtained are essentially an incremental extension of previously published work\nand lack sufficient novelty.\nReply: We thank the reviewer for your comment. While earlier studies have\ndocumented the presence of CHILs across some plant species, our work provides a\nconceptual and mechanistic advance by moving from observational reporting to a\nstructural and functional understanding of CHIL regulation for CHS. Previous work\nlacked the structural basis of the CHS-CHIL complex, which is essential for identifying\nspecific regulatory motifs. Building on this unique structural insight, we achieved\nseveral novel outcomes: we not only identified diverse CHILs but also demonstrated\nfunctional conservation of the β-hairpin mediated regulatory mechanism across land\nplants. Furthermore, by integrating evolutionary analysis with structural information,\nwe rationally designed the CHIL double mutant H36E and F37T, which significantly\nenhances CHS activity, a finding that was made possible only by an integrated analysis\nof structural and evolutionary data. These results allow us to move beyond descriptive\nconservation and establish a conserved \"binding conformational regulation\" paradigm,\nclarifying how CHIL controls metabolic flux at the molecular level. Collectively, this\nstudy transforms what was previously a descriptive evolutionary narrative into a\nstructure informed, predictive framework for engineering flavonoid metabolism.\n5. The manuscript suffers from a lack of experimental detail, which compromises the\nvalidity and reproducibility of the results:\nReply: We are grateful to the reviewer for their constructive feedback, which has\nhelped us improve the precision and quality of our work. We agree that detailed\nmethodology is essential for validating results and ensuring their reproducibility. In\n25 / 39\nresponse, we have provided point-by-point clarifications below and have updated the\nrevised manuscript accordingly.\n· The sources and purity of the chemicals used are not described. Without this critical\ninformation, the experimental results cannot be properly evaluated. For instance, some\ncommercial p-coumaroyl-CoA preparations contain inhibitory impurities that can skew\nresults. To mitigate this, standard CHS assays are often coupled with a 4-\ncoumarate:CoA ligase-catalyzed reaction (see ref 12).\nReply: We thank the reviewer for raising this critical point regarding the sourcing and\npurity of chemicals, and for highlighting the important issue of potential inhibitory\nimpurities in acyl-CoA substrates. We agree that this information is essential for\nevaluating the experimental results, and we have revised the manuscript accordingly.\nThe 'Methods' section has been updated to specify the commercial sources and stated\npurities for all key chemicals. Specifically, p-coumaroyl-CoA (Catalog No. S25276,\npurity ≥90%), naringenin (Catalog No. B21596, purity ≥98%), and naringenin chalcone\n(Catalog No. B21598, purity ≥98%) were purchased from Shanghai YuanYe Bio-\nTechnology Co., Ltd. Malonyl-CoA (Catalog No. M4263, purity ≥90%) was obtained\nfrom Sigma-Aldrich. To mitigate concerns regarding inhibitory impurities, we\nspecifically used this high-purity grade of p-coumaroyl-CoA for all assays. Our\nenzymatic activity assays data demonstrate robust and reproducible CHS activity with\nhigh turnover rates, which is inconsistent with significant inhibition from substrate\nimpurities. The observed activation by CHIL is a consistent and pronounced effect\nacross all replicates, providing confidence in our conclusions. As suggested, we have\nincorporated this information into the Methods section of our revised manuscript.\nFurthermore, as suggested by the reviewer, we conducted CHS activity assays\nusing a 4-coumarate:CoA ligase (4CL)-coupled system (see Fig.R8 below). We\nexpressed and purified 4CL and carried out the coupled assay following the method\ndescribed in ref. 12 (Liou, G. et al., J Biol Chem, 2018). We observed that the amount\nof NAR produced in a two-hour coupled reaction was substantially lower (less than\n26 / 39\n10%) than that generated in a ten-minute assay using direct substrate feeding. We\nattribute this lower efficiency to the inherent complexity of the two-enzyme system and\nits associated procedural requirements, which appear to make the direct provision of\nsubstrates to CHS a more efficient and robust approach for our experimental goals.\nFig.R8 | Extracted ion chromatogram (EIC) of in vitro enzymatic assays products\nwith proteins of the recombinant CHS and 4CL. P1, (2S)-Naringenin; P2, CTAL.\nReferences:\nLiou, G., Chiang, Y. C., Wang, Y. & Weng, J. K. Mechanistic basis for the evolution\nof chalcone synthase catalytic cysteine reactivity in land plants. J Biol Chem 293,\n18601-18612, doi:10.1074/jbc.RA118.005695 (2018).\n· The gel filtration conditions used for the isolation of the CHS-CHIL complex need to\nbe elaborated in detail.\nReply: We thank the reviewer for this suggestion. We have now elaborated on the gel\nfiltration (size-exclusion chromatography) conditions in the revised manuscript. The\ndetailed methodology is as follows: The CHS-CHIL complex was purified using a\nSuperdex 200 Increase 10/300 GL column (Cytiva, Cat No./ID: 28990944) on an\nÄKTA pure system. The chromatography was performed at 4°C in buffer A containing\n20mM Tris-HCl, pH 8.0, and 100mM NaCl. The sample was eluted at a flow rate of\n0.4 mL/min. The elution profile was monitored by measuring the absorbance at 280 nm.\nFractions corresponding to the CHS-CHIL complex peak were collected, concentrated,\nand used for subsequent structural and biochemical studies. These details have been\nadded to the Methods section of the revised manuscript.\n· Please provide CC1/2(%) values in Supplementary Table 1.\nReply: We appreciate you pointing this out. As suggested, the CC1/2 value of 0.997\nhas been incorporated into Supplementary Table 1 in our revised manuscript.\nWe sincerely thank the reviewer for recognizing the structural novelty of the CHS-\nCHIL complex presented in our manuscript, as well as for the other insightful\ncomments. The reviewer’s valuable suggestions have significantly enhanced the rigor\nof our analysis and the clarity of our presentation. We have thoroughly addressed each\nof the raised points and expanded the relevant discussions in the revised version.\nThese inputs have been truly invaluable, and we deeply appreciate the time and thoughtful\nefforts the reviewer has dedicated to our work.\n28 / 39\nReviewer #3 (Remarks to the Author):\nThe authors of this manuscript carried out a comprehensive mechanistic study\naddressing a long-standing question in flavonoid biosynthesis, particularly the function\nof CHIL and the product specificity of CHS (naringenin chalcone vs. the cyclization\nderailment product CTAL) when expressed alone in vitro. Using a combination of\ncrystallography, biochemical assays, and evolutionary analyses, the authors\ndemonstrate that CHIL proteins act as critical non-enzymatic partners that enhance the\nfidelity and efficiency of CHS, preventing the formation of derailment byproducts and\npromoting the specific synthesis of naringenin chalcone, the canonical precursor of\nflavonoids. The integration of in vitro enzyme kinetics, co-expression experiments, and\nmutant analyses confirms that CHIL fine-tunes CHS activity by stabilizing the active\nconformation and channeling intermediates toward the correct product. This work not\nonly clarifies the elusive role of CHIL in flavonoid biosynthesis but also provides\nstructural and mechanistic insights that explain discrepancies observed in earlier studies\nwhere CHS expressed alone yielded multiple non-physiological products. Overall, this\nis an important and well-written paper with well-executed experiments. I provide\ncomments below with the intention of further strengthening and clarifying the paper,\nespecially some of the key mechanistic claims.\nReply: We sincerely thank the reviewer for your positive and encouraging comments.\nYour recognition of our work is greatly appreciated, and your supportive feedback\nmeans a lot to us.\n1. Page 10 “This biochemical evidence confirms an antagonistic relationship between\nCoA and CHIL, explaining our failure to obtain crystals of the CHS-CHIL complex\nwith bound NAR and CoA despite several attempts. These findings suggested that\nCHIL modulates the activity and product specificity of CHS by accelerating CoA\nrelease to increase reaction rates.”\nThis claim seems weak, as the biochemical observations presented earlier in the\n29 / 39\nparagraph could support multiple mechanistic interpretations. It is intriguing that both\nCHS substrates, p-coumaroyl-CoA and malonyl-CoA, contain the large CoA moiety. If\nCHIL binding antagonizes CoA, how does initial p-coumaroyl-CoA binding, as well as\nthe iterative malonyl-CoA binding, occur efficiently? Additionally, it is not specified\nwhether the released CoA originates from p-coumaroyl-CoA or malonyl-CoA.\nI suggest the authors consider performing molecular dynamics (MD) simulations of\nCHS with and without CHIL, across different substrate, intermediate, or product-bound\nstates. Such simulations could substantially enhance the mechanistic understanding of\nhow CHIL modulates the complex catalytic sequence of this iterative enzyme. For\nexample, they might reveal coordinated movements of the β-hairpin relative to the\npolyketide extension, cyclization, and product release steps.\nReply: We appreciate the reviewer’s insightful comments and suggestions. To further\nelucidate the mechanistic basis of CHIL’s modulation of CHS activity, we performed\nmolecular dynamics (MD) simulations of CHS in the presence and absence of CHIL\nacross three representative binding states: substrate-bound (p-coumaroyl-CoA or\nmalonyl-CoA) and product-bound (NAR and CoA).\nThe simulations revealed that CHIL binding significantly stabilizes substrate-\nbound states, as indicated by smaller RMSD, narrower RMSD distributions, and\nreduced fluctuations in the internal angle for p-coumaroyl-CoA and malonyl-CoA\ncompared with the CHIL-free systems (Extended Data Fig. 14 and 15). In contrast,\nCHIL destabilizes the product-bound CoA, which exhibits larger RMSD values,\nbroader fluctuation ranges, and more variable internal angle changes (Extended Data\nFig. 15). Furthermore, we observed that the -hairpin of CHIL frequently interacts with\nCoA thorough hydrogen bonds but rarely with p-coumaroyl-CoA and almost never with\nmalonyl-CoA.\nCombining these computational findings with our biochemical data, we propose\nthat CHIL enhances CHS catalysis through a dual mechanism: (1) Stabilizing substrate\nbinding to maintain a productive catalytic conformation during the reaction cycle;\n(2)\n30 / 39\nDestabilizing the product-CoA complex through the interaction with CHIL, thereby\nfacilitating CoA release after product formation and increasing overall turnover.\nExtended Data Fig. 14 | Ligand stability in substrate- and product-bound CHS\nsystems with and without CHIL.\na, Violin plots showing the RMSD distributions of p-coumaroyl-CoA, malonyl-CoA,\nNAR, and CoA over 200 ns MD simulations each with three replicas. RMSD values\nwere computed after aligning the protein backbone to the initial structure. The upper\nand lower blue lines indicate the maximum and minimum RMSD values, while the\ngreen and orange lines represent mean and median RMSD values, respectively. b, The\nangle definition by the two lines formed by the two groups of atoms in CoA moiety\nhighlighted as sphere (P - O and N - N). The line plots showing the angle changes along\n31 / 39\ntime for p-coumaroyl-CoA, malonyl-CoA and CoA at the absent (left column) or the\npresence (right column) of CHIL. Replicas 1, 2, and 3 are colored with blue, orange,\nand green, respectively.\nExtended Data Fig. 15 | Conformational changes of β-hairpin in CHIL and the\nsnapshots of CHIL relative to CHS and bound substates or CoA.\na, The motion of β-hairpin in CHIL along time, which is quantified by the angular\nchanges of the direction from the Cα atom of T31 to that of I35 relative to the initial\ndirection. b, The snapshots after 200 ns simulations at the presence of CHIL. Each\ncolumn refers to one of binding states. The interaction of CoA and E34 or F37 is\nhighlighted.\n32 / 39\nWe have revised the paragraph accordingly in the main text:\n“Following the biochemical experiments, we performed conventional molecular\ndynamics (MD) simulations to further investigate how CHIL modulates CHS activity\nacross different catalytic stages. We examined three representative binding states of\nCHS in the presence and absence of CHIL: substrate-bound (p-coumaroyl-CoA or\nmalonyl-CoA) and product-bound (NAR and CoA). To quantitatively assess the\nstability of substrates and products within the CHS binding pocket, we calculated the\nRMSD of the ligand heavy atoms after aligning to the initial protein structures. In the\nsubstrate-bound simulations, p-coumaroyl-CoA exhibited an average RMSD of 4.0 Å\n(median: 4.1 Å, standard deviation (std): 0.7 Å) in the presence of CHIL, compared\nwith 5.6 Å (median: 5.4 Å, std: 1.9 Å) in the CHIL-free systems. Similarly, malonyl-\nCoA exhibited an average RMSD of 4.5 Å (median: 3.8 Å, std: 2.0 Å) with the presence\nof CHIL, versus 6.2 Å (median: 6.2 Å, std: 2.1 Å) without CHIL (Extended Data Fig.\n14). By calculating the angle of CoA moiety of the substates (Extended Data Fig. 14b),\nwe observed that the fluctuation of the angle of p-coumaroyl-CoA (std: 24.1°) or\nmalonyl-CoA (std: 32.9°) is markedly lower than those (std: 39.6° and 40.9°,\nrespectively) in the CHIL-free systems. This indicates that CHIL restricts the\nconformational flexibility of bound substrates. The NAR exhibited very stable in both\ncases with the averaged RMSD of 2.5 Å (median: 2.4 Å, std: 0.8 Å) at the present of\nCHIL, compared with 2.3 Å (median: 2.2 Å, std: 0.3 Å) in the absent of CHIL. Except\nNAR, the lower RMSD values and narrower RMSD fluctuation in the CHIL-bound\nsystems indicate that CHIL can enhance the occupancy of substrates in the binding\npocket. In the product-bound simulations, CoA displayed an average RMSD of 6.5 Å\n(median: 6.3 Å, std: 2.2 Å) in the presence of CHIL, compared with 4.2 Å (median: 4.5\nÅ, std: 1.2 Å) in the absence of CHIL (Extended Data Fig. 14). The CoA moiety exhibits\npronounced angular fluctuations between 40° and 96° in replicas 1 and 3, whereas in\nthe absence of CHIL the angles remain relatively stable-around 47° in replica 3 and\n123° in replicas 1 and 2 (Extended Data Fig. 14b). These observations suggest that\nCHIL can make substrates more stable in their binding pockets and CoA less stable,\n33 / 39\nwhich may contribute the occurrence of catalytic reaction of substates and the release\nof CoA.\nWe further analyzed the motion of the CHIL’s β-hairpin by calculating the angle\ndefined by the direction vector from the Cα atom of T31 to that of I35 relative to its\ninitial orientation. We observed that the β-hairpin can rotated up to 60° in the three\nbinding states and the orientation of the β-hairpin ends with ~45° degree in most of 200\nns simulations (6/9) (Extended Data Fig. 15). Notably, in replica 1 of the CoA-bound\nsystem, the -hairpin adopts a much more stable conformation centered around ~17°\n(Extended Data Fig. 15). This stability correlates with hydrogen bonds formed between\nthe terminal nitrogen of CoA and the carboxylate oxygens of E34 (occupancy: 47.0%)\nor the backbone oxygen of F37 (occupancy: 5.9%) (Extended Data Fig. 15),\nsuggesting\nthat CoA directly interact the β-hairpin in this replica. By contrast, CHIL forms only\nlimited interactions with p-coumaroyl-CoA and almost none with malonyl-CoA. The\nmarkedly higher frequency of CoA-CHIL interactions, together with the pronounced\nfluctuations of CoA in CHIL-bound simulations (Extended Data Fig. 15), indicates\nincreased CoA mobility. This enhanced mobility may facilitate its release from the\nactive site. Together, these observations suggest that CHIL can promote catalysis by\nstabilizing substrates and facilitating product release, thereby accelerating the overall\nreaction cycle.”\nWe also have added the following paragraph in the method text:\n“Molecular dynamics (MD) simulations\nTo further elucidate how CHIL affects different stages of the CHS catalytic process, we\nperformed MD simulations on six systems corresponding to three representative\nbinding states with or without CHIL: substrate-bound (p-coumaroyl-CoA or malonyl-\nCoA) and product-bound (NAR and CoA). Initial complex structures were prepared as\nfollows. For CHS, we used the experimentally determined crystal structures available\nfrom this work (PDB ID: 9WBE) to ensure accurate active site geometry. For the\n34 / 39\nproduct-bound ligands NAR and CoA, binding poses were extracted from a previously\nresolved CHS-product complex (PDB ID: 7VF0) to maintain experimentally validated\nligand positions. For other components (CHIL, p-coumaroyl-CoA, and malonyl-CoA),\nwe used AlphaFold31 to predict the dimeric CHS-CHIL assemblies following the\nalignment to the CHS crystal structure for their coordinates. The predicted CHIL\nstructure was used instead of the crystallographic model to avoid steric clashes with\nsubstrates or CoA observed in preliminary modeling. All systems were constructed\nusing CHARMM-GUI2,3 with the CHARMM36m4 force field for proteins and\nCGenFF5 for small molecules. Protonation states were assigned at pH 7.0 using\nPROPKA6. Systems were solvated with TIP3P water7 and neutralized with 0.15 M\nNaCl. The simulation boxes measured approximately 97 x 89 x 73 Å3 (without CHIL)\nor 121 x 100 x 91 Å3 (with CHIL), containing ~59,000 or ~104,000 atoms, respectively.\nEnergy minimization was carried out using steepest-descent and adopted-basis\nNewton-Raphson algorithms8 (50 steps each). Solvent relaxation was performed under\nNVT conditions for 5 ps with restraint force constant of 400.0 kJ/mol/nm2 on protein\nbackbone and small molecule heavy atoms and that of 40.0 kJ/mol/nm2 on protein\nsidechain atoms. Production simulations were conducted using OpenMM9 at 303.15 K\nand 1 bar with a 2 fs timestep. Long-range electrostatics were computed using the\nparticle-mesh Ewald method10 with a 12 Å real-space cutoff, and Lennard-Jones\ninteractions were smoothed between 10 Å and 12 Å. Hydrogen-involving bonds were\nconstrained with SHAKE11. Each simulation was run for 200 ns and repeated three\ntimes with different random seeds. Trajectory analyses, including RMSD and ligand\nmovement quantification, were performed using MDAnalysis12.”\nReferences:\n3. Nature 630, 493-500 (2024). https://doi.org/10.1038/s41586-024-07487-w\nCHARMM. Journal of Computational Chemistry 29, 1859-1865 (2008).\nhttps://doi.org/https://doi.org/10.1002/jcc.20945\n35 / 39\nsimulations. Journal of Computational Chemistry 35, 1997-2004 (2014).\nhttps://doi.org/https://doi.org/10.1002/jcc.23702\nproteins. Nature Methods 14, 71-73 (2017). https://doi.org/10.1038/nmeth.4067\ncompatible with the CHARMM all-atom additive biological force fields. Journal of\nComputational Chemistry 31, 671-690 (2010). https://doi.org/https://doi.org/10.1002/jcc.21367\nTreatment of Internal and Surface Residues in Empirical pKa Predictions. Journal of Chemical\nTheory and Computation 7, 525-537 (2011). https://doi.org/10.1021/ct100578z\nof simple potential functions for simulating liquid water. The Journal of Chemical Physics 79,\n926-935 (1983). https://doi.org/10.1063/1.445869\n551 (1995). https://doi.org/10.1137/1037125\nmolecular dynamics. PLOS Computational Biology 13, e1005659 (2017).\nhttps://doi.org/10.1371/journal.pcbi.1005659\n103, 8577-8593 (1995). https://doi.org/10.1063/1.470117\n11 van Gunsteren, W. F. & Berendsen, H. J. C. Algorithms for macromolecular dynamics and\nconstraint dynamics. Molecular Physics 34, 1311-1327 (1977).\nhttps://doi.org/10.1080/00268977700102571\nthe analysis of molecular dynamics simulations. Journal of Computational Chemistry 32, 2319-\n2327 (2011). https://doi.org/https://doi.org/10.1002/jcc.21787\n2. It remains unclear how the balance between the naringenin chalcone product and the\nCTAL derailment product is regulated in the presence or absence of CHIL. Insights into\nthis could also potentially be gained through MD simulations.\nReply: We thank the reviewer for this valuable suggestion. To further investigate how\nCHIL modulates the balance between canonical products (NC and NAR) and\nderailment byproducts (BNY and CTAL), we performed MD simulations on four CHS-CHIL complexes, each bound with a single ligand. The ligands exhibited comparable\naverage RMSD values (CTAL: 4.1 Å, BNY: 3.4 Å, NC: 3.0 Å, NAR: 3.4 Å), indicating\nthat all molecules remained close to their initial binding poses (Extended Data Fig. 16).\nNotably, CTAL displayed two distinct RMSD peaks (3.25 Å and 5.86 Å), suggesting\nthe presence of two alternative binding positions. Although CTAL was relatively less\nstable, these results indicate that CHIL does not significantly alter the stability of the\nfinal bound products, implying that its regulatory effect likely occurs at earlier stages\nof the catalytic cycle, such as substrate binding or intermediate channeling.\nExtended Data Fig. 16 | Violin plots showing the RMSD distributions of the bound\nbyproducts (CTAL and BNY) and products (NC and NAR) over 200 ns MD\nsimulations of CHS-CHIL complexes.\nRMSD values were calculated after alignment of the protein backbone to the initial\nstructure. The upper and lower blue lines indicate maximum and minimum RMSD\nvalues, while the green and orange lines represent mean and median RMSD values,\nrespectively.\nWe have revised the paragraph accordingly in the main text:\n“We further performed MD simulations to investigate whether CHIL differentially\nstabilizes canonical products (NC and NAR) versus derailment by-products (BNY and\nCTAL). Four CHS-CHIL systems were constructed, each bound with a single ligand.\nThe ligands maintained binding poses close to their initial positions, as reflected by\ntheir average RMSD values (CTAL: 4.1 Å, BNY: 3.4 Å, NC: 3.0 Å, NAR: 3.4 Å)\n(Extended Data Fig. 16). Notably, CTAL displayed two distinct RMSD peaks (3.25 Å\n37 / 39\nand 5.86 Å), suggesting the existence of two alternative binding positions (Extended\nData Fig. 16). Although CTAL was relatively less stable, these results indicate that\nCHIL does not significantly alter the stability of the final bound products, implying that\nits regulatory effect likely occurs at earlier stages of the catalytic cycle, such as substrate\nbinding or intermediates.”\nWe also have added the following paragraph in the method text:\n“In addition, to examine the potential role of CHIL in balancing the formation of\nnaringenin chalcone versus derailment products, we constructed four additional CHS-\nCHIL systems bound with either the canonical products (NC and NAR) or the\nbyproducts (BNY and CTAL). The CHS and CHIL structures were obtained from this\nstudy, while the bound small molecules were modeled using AlphaFold3 predictions\n(except NAR, taken from PDB ID: 7VF0). System preparation, force field parameters,\nand simulation protocols were identical to those described above.”\n3. Did the authors observe smaller derailment products from single- or double-\ncondensation events in addition to CTAL? If so, were these byproducts reduced in the\npresence of CHIL?\nReply: We appreciate the reviewer's insightful comment. Following your suggestion,\nwe carefully re-examined our Mass Spectrometry data for enzyme activity assays. Our\nanalysis showed that, regardless of whether the Total Ion Chromatogram (TIC) or\nspecific Extracted Ion Chromatograms (EICs) were used (e.g., for BNY at m/z=229),\nno minor derailment products besides CTAL were observable. As documented in the\nliterature (Waki et al., Nat Commun, 2020), BNY is only produced in minimal amounts\nin the CHS-catalyzed reaction, which could explain why its signal falls below the\ndetection limit in our assay.\nThe exact quotation from the referenced article is as follows: “Our assay results\nrevealed that the product ratios of the CHS-catalyzed reactions varied with the enzyme\nsource, with the formation of BNY being negligible in all cases (<1% (mol/mol) of all\n38 / 39\nproducts).”\nReferences:\nWaki, T. et al. A conserved strategy of chalcone isomerase-like protein to rectify\npromiscuous chalcone synthase specificity. Nat Commun 11, 870 (2020).\n4. Please ensure that ITF1 and ITF2 are properly defined upon first mention. What do\nthese abbreviations stand for?\nReply: We appreciate this valuable suggestion. We have clarified in the revised\nmanuscript that ITF1 and ITF2 are abbreviations for Interface 1 and Interface 2, which\nrepresent the two primary contact surfaces between CHS and CHIL in the complex.\nThe relevant sections have been updated accordingly.\nWe sincerely thank the reviewer for the generous and encouraging assessment of\nour manuscript. We are grateful for the recognition of our experimental design, clear\nwriting, and the significance of the findings. The reviewer's insightful comments have\ngreatly improved the rigor of our analysis and the clarity of our presentation. We have\ncarefully addressed all comments and expanded the discussions in the revision. These\ninputs have been invaluable, and we sincerely appreciate the reviewer's time and\nthoughtful efforts.\n39 / 39"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed many of the requested items.\nIt would be appreciated if the authors could further address the items below.\nItem 5: ITC methods describe that at least 3 experiments were performed per sample. This is not reflected in the single Kd values presented in Ext. data Fig. 8. The Kd values should represent the mean value for the replicate experiments +/- error.\nItem 8: In figure 1D, the authors present a new SDS-PAGE gel corresponding to the chromatogram, presumably in order to address the CHIL dimer question. I find it puzzling that the chromatogram is identical to the one presented in the last version of the manuscript, although the old and new gels show very different protein content in the respective elution fractions. To illustrate this, I've created a simple overlay of the two versions of figure 1d. It's unclear to me how such different protein samples (shown in gels) can create identical chromatograms. Some clarification would be helpful.\n[Editorial Note: Please also see attachment at end of file]", "reviewer_2": "I have evaluated the revised manuscript, focusing primarily on the authors' responses to my previous concerns. While the revision includes new data, several critical issues remain.\n1. Major Concern: Discrepancy in Kinetic Parameters\nThere remains a significant concern regarding the reported kcat values. In plant specialized metabolism, enzymes like\nchalcone synthase (CHS) generally exhibit low turnover rates—typically less than 1/30th of the kcat seen in central primary\nmetabolism (Bar-Even A., et al., Biochemistry 50, 4402 (2011)). For context, alfalfa CHS (without CHIL) has a reported kcat\nof approximately 0.1–0.3 s−1 (Jez and Noel, J. Biol. Chem.2000), and other species typically report values between 0.01\nand 0.1 s−1 (Waki et al. Nature Commun. 11, 870 (2020)). In contrast, the authors report values approaching 1000 s−1. This\nis more than 10,000-fold higher than established values for plant CHSs and even exceeds the efficiency of most primary\nmetabolic enzymes. The authors must rigorously justify the validity of these kinetic parameters, as such an extreme outlier\nrequires extraordinary evidence.\n2. Molecular Dynamics (MD) Simulations\nThe inclusion of MD simulations in the revised version provides valuable insight into how CHIL enhances CHS activity.\nHowever, these analyses do not appear to clarify how CHIL regulates CHS specificity. While this may be a limitation of the\ncurrent study, it would be beneficial to address this in the Abstract to provide a more balanced overview. I suggest adding a\nsentence such as that described by the authors themselves:\n\"MD simulation results suggest that the regulatory effect of CHIL on CHS specificity likely occurs at earlier stages of the\ncatalytic cycle, such as during substrate binding or the formation of early intermediates.\"", "reviewer_3": "The authors have thoroughly addressed my previous comments. I have no additional comments."}, "author_response": "Point-by-point response to the referees’ comments\nDear Editor and Referees,\nWe appreciate your valuable comments on the manuscript (NCOMMS-25-\n61179B). Following thorough consideration of each comment and suggestion, we have\ndiligently addressed them point-by-point in the revision outlined below.\nReviewer #1 (Remarks to the Author):\nThe authors have adequately addressed many of the requested items. It would be\nappreciated if the authors could further address the items below.\nReply: Thank you for your positive feedback and for the additional suggestions. We\nappreciate the opportunity to further improve our manuscript and will carefully address\neach of the points below.\nItem 5: ITC methods describe that at least 3 experiments were performed per sample.\nThis is not reflected in the single Kd values presented in Ext. data Fig. 8. The Kd values\nshould represent the mean value for the replicate experiments +/- error.\nReply: Thank you for your comments. We sincerely apologize for the oversight in\npresenting single K values instead of the mean and error from our replicate ITC\nD\nexperiments. The revised manuscript now presents the data in Extended Data Fig. 8 as\nthe mean ±SD from three independent measurements, and the associated text has been\nupdated accordingly. Three independent ITC measurements yielded the following K\nD\nvalues: for the CHIL WT protein, 7.35, 7.58, and 8.47 M; for the H36L mutant, 1.01,\n1.20, and 1.58 M; and for the H36A mutant, 10.84, 11.00, and 13.30 M.\n1 / 8\nExtended Data Fig. 8 | ITC measurement of CHS binding affinity to CHIL and its\nmutants. Titration curves for CHIL (wild-type) and the mutants (H36L, H36A, F37A,\nM4A, K97A, Q100A, M3A) are colored in red, purple, blue, green, cyan, brown, yellow,\nand gray, respectively.\nItem 8: In figure 1D, the authors present a new SDS-PAGE gel corresponding to the\nchromatogram, presumably in order to address the CHIL dimer question. I find it\npuzzling that the chromatogram is identical to the one presented in the last version of\nthe manuscript, although the old and new gels show very different protein content in\nthe respective elution fractions. To illustrate this, I've created a simple overlay of the\ntwo versions of figure 1d. It's unclear to me how such different protein samples (shown\nin gels) can create identical chromatograms. Some clarification would be helpful.\nReply: We sincerely thank Reviewer for their careful evaluation and for raising this\nimportant concern regarding the apparent discrepancy between the gel filtration\nchromatogram and the SDS-PAGE results in Figure 1D across manuscript versions. We\nappreciate the opportunity to clarify this point in detail.\n2 / 8\nThe observed differences between the original and revised SDS-PAGE gels are\nunderstandable and do not reflect an inconsistency in the underlying chromatography\ndata. Our explanation is as follows:\n1. The gel filtration experiment was completed in August 2022. The SDS-PAGE gel in\nour initial submission was prepared from freshly collected fractions at that time. The\ngel presented in the revised manuscript was run in September 2025 using aliquots from\nthe same original purification, which had been stored at -80°C for over three years.\nProlonged storage can lead to gradual sample degradation or aggregation within stored\nfractions. Additionally, minor technical variations in gel concentration, sample loading,\nor electrophoresis conditions between the two time points can affect band appearance\nand intensity. Critically, however, the central finding remains unambiguous: the bands\ncorresponding to the CHS-CHIL complex in the first peak is clearly present and\nconsistent across both gels. This finding robustly validates the stable interaction\nbetween CHS and CHIL, a key conclusion drawn from the present experiment.\nPrecisely this complex, isolated from the first peak, was successfully crystallized for\nstructural determination.\n2. The original stored fractions were limited and were exhausted. To directly address\nany concerns about reproducibility, we have since performed three independent\npurifications of the CHS-CHIL complex (Fig. R1 below). These results consistently\nconfirm the stable formation of the complex. We consistently observe two main peaks.\nSince the complex is purified via the 6xHis-tag on CHIL (CHS is untagged), the precise\nratio of the peaks can vary between purifications, as is common with affinity-based\nmethods. Nevertheless, the presence of the stable CHS-CHIL complex in the first peak\nis a reproducible and invariant outcome.\n3 / 8\nFig. R1 | Size-exclusion chromatograms and Coomassie blue-stained SDS-PAGE gels\nof the CHS-CHIL complex from three independent experiments.\n3. To eliminate any ambiguity and present the most consistent data, we have prepared\na new, unified Figure 1D. This figure pairs a representative chromatogram from our\nreproducible purifications with an SDS-PAGE analysis of freshly collected fractions\nfrom that same run, ensuring perfect correspondence. We believe this updated figure\nwill fully resolve the concern.\nFig.1 | The function of CHS and CHIL.\n4 / 8\na, Flavonoid biosynthesis pathway involving CHS and CHIL. PAL, phenylalanine\nammonia lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate-CoA ligase;\nCHS, chalcone synthase; CHIL, chalcone isomerase-like; CHI, chalcone isomerase. b,\nExtracted ion chromatogram (EIC) of in vitro enzymatic assays products with proteins\nof the recombinant CHS and CHIL. P1, (2S)-Naringenin; P2, CTAL; NAR-STD, (2S)-\nNaringenin authentic standard. c, SDS-PAGE illustrating the binding ability between\nCHS with CHI and CHIL determined through pull-down experiments. CHS was fused\nto glutathione S-transferase (GST-tag), and CHIL and CHI were fused to 6xHis-tag as\nthe bait. d, Size-exclusion chromatogram and Coomassie-blue stained SDS-PAGE of\nCHS-CHIL complex. e, In vitro enzymatic assays between CHS with CHIL or CHI. “-”\nrepresents not adding CHS for the enzymatic assays. Letters indicate significant\ndifferences determined by one-way ANOVA, p < 0.05, n≥3. Error bars indicate ±SD.\nWe are truly grateful for the insightful comments and considerable effort you\ninvested in reviewing our manuscript. Your thoughtful suggestions guided our revisions\nand were crucial to refining the work. We have addressed each of the concerns you\nraised and sincerely thank you for your help in improving this study.\n5 / 8\nReviewer #2 (Remarks to the Author):\nI have evaluated the revised manuscript, focusing primarily on the authors' responses\nto my previous concerns. While the revision includes new data, several critical issues\nremain.\nReply: We are extremely grateful for your thorough review and for pointing out the\ncritical issues that still need attention. We will address each of the remaining points\nbelow and have revised the manuscript accordingly.\n1. Major Concern: Discrepancy in Kinetic Parameters\nThere remains a significant concern regarding the reported k values.\ncat\nIn plant specialized metabolism, enzymes like chalcone synthase (CHS) generally exhibit\nlow turnover rates-typically less than 1/30th of the k seen in central primary\ncat\nmetabolism (Bar-Even A., et al., Biochemistry 50, 4402 (2011)). For context, alfalfa\nCHS (without CHIL) has a reported k of approximately 0.1-0.3 s-1 (Jez and Noel, J.\ncat\nBiol. Chem.2000), and other species typically report values between 0.01 and 0.1 s-1\n(Waki et al. Nature Commun. 11, 870 (2020)). In contrast, the authors report values\napproaching 1000 s-1. This is more than 10,000-fold higher than established values for\nplant CHSs and even exceeds the efficiency of most primary metabolic enzymes. The\nauthors must rigorously justify the validity of these kinetic parameters, as such an\nextreme outlier requires extraordinary evidence.\nReply: We sincerely thank you for your professional and valuable comments. In\nresponse, we conducted a rigorous re-evaluation of the kinetic parameters, which\nincluded recalculating the original data ourselves and having the calculations verified\nby two independent experts proficient in enzyme kinetics. This process confirmed that\nthe values of K and V were accurate. Additionally, we repeated the enzyme activity\nm max\nassays for a subset of samples, which showed good reproducibility, further validating\nthe robustness of our original experimental data. The discrepancy in the k calculation\ncat\narose from a unit conversion error related to the enzyme concentration. Specifically, we\n6 / 8\nmistakenly used the enzyme mass (0.0025 mg) as the concentration to calculate k ,\ncat\nwithout converting it to molar concentration. We have now corrected this mistake and\nupdated all relevant parameters in the corresponding table accordingly. The revised\ntable is provided below for your reference (Supplementary Table 2). For further details,\nplease refer to Supplementary Table 5. We sincerely appreciate your thorough and\ninsightful review, which has been invaluable in improving the quality of our manuscript.\nSupplementary Table 2. Kinetic parameters of CHS-catalyzed NAR production\nwith malonyl-CoAa and p-coumaroyl-CoAb in the absence and presence of CHIL\nvariants. a50 M p-coumaroyl-CoA was used as the starter substrate,b100 M malonyl-\nCoA was used as the extender substrate.\n2. Molecular Dynamics (MD) Simulations\nThe inclusion of MD simulations in the revised version provides valuable insight into\nhow CHIL enhances CHS activity. However, these analyses do not appear to clarify\nhow CHIL regulates CHS specificity. While this may be a limitation of the current study,\nit would be beneficial to address this in the Abstract to provide a more balanced\noverview. I suggest adding a sentence such as that described by the authors themselves:\n\"MD simulation results suggest that the regulatory effect of CHIL on CHS specificity\nlikely occurs at earlier stages of the catalytic cycle, such as during substrate binding or\nthe formation of early intermediates.\"\nReply: We appreciate the reviewer's suggestion to clarify the results of the MD\n7 / 8\nsimulations. Accordingly, we have incorporated the following sentence into the\nAbstract as recommended: \"MD simulation results suggest that the regulatory effect of\nCHIL on CHS specificity likely occurs at earlier stages of the catalytic cycle, such as\nduring substrate binding or the formation of early intermediates.\"\nWe are grateful to you for your meticulous assessment of our manuscript and your\nother insightful suggestions. Your valuable input has substantially improved the rigor\nof our analysis and the clarity of the text. All raised points have been addressed on a\npoint-by-point basis. We sincerely appreciate the considerable time and expertise you\nhave invested in this review.\nReviewer #3 (Remarks to the Author):\nThe authors have thoroughly addressed my previous comments. I have no additional\ncomments.\nReply: Thank you very much for your final confirmation and for your time throughout\nthe review process. We are pleased to hear that our revision has fully addressed your\nprevious comments. We appreciate your valuable contributions to improving our\nmanuscript.\n8 / 8"}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed my concerns.", "reviewer_2": "Discrepancies in the initial calculations have been resolved. The revised kcat values are now in agreement with literature standards. I am satisfied with these revisions and have no additional comments."}, "author_response": ""}]

Decision letter