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. 2025 May 15;474:143077. doi: 10.1016/j.foodchem.2025.143077

Unraveling the trisubstrate-triproduct reaction mechanisms of wine grape VvCYP76F14 to improve wine bouquet

Zhizhong Song a,b,c,1, Meiling Tang c,d,e,1, Huilin Xiao c,d,e, Houhua Xu c,e, Matthew Shi b,c, Adeeba Dark b, Zhenqiang Xie a,e,f,⁎, Bin Peng a,c,f,⁎
PMCID: PMC11877275  PMID: 39893722

Abstract

Bouquet is a captivating wine characteristic that affects consumer consumption. Wine lactone correlates with both precursor ((E)-8-carboxylinalool) concentration and wine age that has the greatest contribution to bouquet. In wine grape, cytochrome P450 VvCYP76F14 can catalyze trisubstrate-triproduct reaction processes (hydroxylation, dehydrogenation and carboxylation) to produce (E)-8-carboxylinalool. However, the exact mechanism of the whole reaction is unclear. Here, we unraveled the multi-catalysis mechanism with the aim of rapid conversion of linalool into (E)-8-carboxylinalool in winemaking. Results showed that the redox partner NADPH cytochrome P450 reductase (VvCPR1) was indispensable for hydroxylation and carboxylation, but not dehydrogenation oxidation. Furthermore, the VvCYP76F14-VvCPR1 complex was introduced in the aging stage of winemaking and results showed that the complex could improve the bouquet by increasing the content of wine lactone and shortening the aging time. Nonetheless, this study reveals the trisubstrate-triproduct reaction mechanism of VvCYP76F14 and the VvCYP76F14/VvCPR1 complex has the potential use for wine bouquet enrichment.

Keywords: Wine grape, Wine bouquet, Cytochrome P450 enzyme, Trisubstrate-triproduct, Catalytic mechanisms

Highlights

  • •

    VvCYP76F14 can catalyze trisubstrate-triproduct reactions.

  • •

    CYP76F14-(E)-8-hydroxylinalool reaction is the rate-limiting step.

  • •

    VvCPR1 was indispensable for hydroxylation and carboxylation.

  • •

    D299T mutation caused loss of (E)-8-oxolinalool and (E)-8-carboxylinalool in vitro.

  • •

    VvCYP76F14-VvCPR1 complex improved wine lactone during aging.

1. Introduction

Wine bouquet is an essential indicator of the sensory characteristics and overall quality of wine, garnering significant interest in the realm of wine consumption (Alegre et al., 2020; Thomas-Danguin et al., 2011; Zhai et al., 2023). The aroma of wine primarily originates from two sources. One source is the flavor compounds present in wine grape berries, which contribute sensory attributes such as floral, sweet, herbal, and fruit notes, collectively referred to as primary aromas (Alegre et al., 2020; Noguerol-Pato et al., 2012). The other source involves the emergence of new compounds, known as wine bouquet or secondary aromas, which develop from flavor precursors during the biochemical processes of fermentation and aging (Wang et al., 2017; Zhai et al., 2023). While primary aromas tend to diminish gradually in wine during the aging process, the wine bouquet plays a pivotal role in imparting the distinctive aroma characteristics to the wine (De la Calle García et al., 1997; Parker et al., 2017; Thomas-Danguin et al., 2011).

During the past decades, researchers have identified hundreds of compounds responsible for wine bouquet (Alegre et al., 2020; Lin et al., 2019). However, only a relatively limited number of compounds, such as sesquiterpenes, fusel alcohols, esters, and lactones, have been found to play crucial roles in creating the typical wine bouquet (Alegre et al., 2020). Notably, the significance of bicyclic monoterpene lactones (wine lactone) in shaping wine bouquet has been underscored in numerous studies, owing to their pleasant aroma and extremely low odor threshold.

It is important to note that wine lactone is not naturally formed in grape berries. Instead, it is generated during the aging of wine, derived from the essential precursor (E)-8-carboxylinalool (Giaccio et al., 2011; Ilc et al., 2017). Wine lactone is correlated with both the age of the wine and the concentration of (E)-8-carboxylinalool (Ilc et al., 2017). Accordingly, shortening the aging time or increasing the content of (E)-8-carboxylinalool is essential to improve the wine's bouquet. In recent years, commercial enzymes have been used to enhance wine quality during various winemaking steps (Francisco, 2020; Michlmayr et al., 2010; Palomo et al., 2005). According to International Vine and Wine Organization (OIV), only a few commercial enzymes are used during wine stabilization and filtration, such as β-glucanase, pectinase, β-glucosidase, urease, and lysozyme. However, limited information is available regarding enzymes addition during the aging process.

The biosynthesis of (E)-8-carboxylinalool in grape berries involves a three-step enzymatic process catalyzed by the trisubstrate-triproduct enzyme VvCYP76F14, a Cytochrome P450 enzyme found in grapevine (Ilc et al., 2017; Lin et al., 2019; Peng et al., 2024). First, VvCYP76F14 catalyzes the hydroxylation of linalool at C8, resulting in the formation of (E)-8-hydroxylinalool. Subsequently, (E)-8-hydroxylinalool undergoes dehydrogenation oxidation at the same carbon atom, leading to the production of (E)-8-oxolinalool. Finally, (E)-8-oxolinalool is transformed into (E)-8-carboxylinalool through carboxylation. In contrast to the majority of plant monofunctional P450s, which typically catalyze a single substrate, the CYP76 family stands out for its multifunctional monooxygenase ability to catalyze multiple substrates (Lin et al., 2019; Paine et al., 2005). In Arabidopsis thaliana, AtCYP76C1 can convert linalool into several compounds, including 8-hydroxylinalool, 8-oxolinalool, and 8-carboxyllinalool, as well as produce lilac aldehydes and lilac alcohols (Boachon et al., 2015). Notably, CYP76 belongs to the Class II monooxygenase P450 system, and the redox partner NADPH cytochrome P450 reductase (CPR) acts as the electron donor, transferring electrons from NADPH to the P450 enzyme, enabling its catalytic activity (Jensen & Møller, 2010; Urban et al., 1997; Werck-Reichhart & Feyereisen, 2000; Zhang et al., 2020). The Class II monooxygenase region of the CYP450s is highly conserved and features the general active motif signature A(G)G(A)XD(E)T (Hofer et al., 2014; Renault et al., 2014). However, the mechanism underlying the complete enzymatic cycle of trisubstrate-triproduct enzyme, including how it effectively manages multi-product release for efficient catalysis, remains largely unknown. In particular, the question of whether CPR is implicated in the catalytic process of multiple substrates is a critical issue that requires urgent resolution.

In this study, VvCYP76F14 was isolated from the traditional wine grape cultivar ‘Heihuxiang’ (Vitis vinifera × V. amurensis) to investigate the underlying catalytic mechanisms. Computer modelling, molecular docking, molecular dynamics calculations, subcellular localization, in vitro expression in Escherichia coli, and co-immunoprecipitation (Co-IP) were used to elucidate the trisubstrate-triproduct reaction mechanism of VvCYP76F14. An evaluation has been made of the enzyme complex in enhancing the potential bouquet of wines.

2. Materials and methods

2.1. Chemicals

In this study, the synthesized and purified forms of (E)-8-hydroxylinalool, (E)-8-oxolinalool, (E)-8-carboxylinalool and wine lactone were obtained from Accela ChemBio Co. Ltd. (Shanghai, China), as described by Peng et al. (2024). The key precursor of linalool was purchased from J and K Scientific Co. Ltd. (Shanghai, China).

2.2. Wine grape and wine samples

In this study, the self-rooted American species of wine grape cultivar ‘Heihuxiang’, a cross between V. vinifera and V. labrusca, was used throughout the entire study (Peng et al., 2024). Similar maturity berries of ‘Heihuxiang’ were collected from the National Grape Germplasm Repository in Yantai (China) at 105 days after full bloom (DAFB). The collected berries were promptly transported to the laboratory within 30 min after harvesting, then chopped, frozen in liquid nitrogen, and stored at −80 °C Ultra-low Temperature Freezer. Three biological replicates were conducted, with each replicate consisting of 20 individual clusters.

Wine samples used in this study were brewed by the Shandong Technology Innovation Center of Wine Grape and Wine/ COFCO Great Wall Wine (Penglai) Co. Ltd. located in Yantai, China, using the standard specification for small container inoculation fermentation brewing to guarantee the uniformity across the wine's production (Ling et al., 2022; Xiao et al., 2020). After being harvested, the stems of wine grape berries were removed and crushed, and placed in a 5 L glass jar. Potassium metabisulfite 0.12 g∙L−1 and commercial brewing yeast CECA 0.3 g∙L−1 were added, and small container fermentation was carried out at 20–25 °C for 10 days. The weight of the liquor was below 0.997 and remained unchanged. The juice residue was separated, and then the self-flowing liquor and pressed liquor were mixed. The alcohol content of the young wine was 12.5 %, pH value was around 4.0, the free sulfur dioxide was maintained at 45 mg∙L−1, and the total acidity of the finished wine was around 5.3 g∙L−1. After the liquor was clarified, the supernatant was taken for full bottle aging, which was stored in the dark at 18–20 °C. Wine samples were collected from the same vineyard and wine quality was further conducted.

2.3. Extraction and quantification of linalool and derivatives

In this study, 31 wine grape varieties with the same berry maturity stage were carefully chosen, and the concentration of linalool and derivatives (including (E)-8-hydroxylinalool, (E)-8-oxolinalool, and (E)-8-carboxylinalool) in the grape berries were determined by Thermo Fisher Scientific Inc. (Shanghai China), using high-performance liquid chromatography combined with high-resolution mass spectrometry (HPLC-HRMS, Thermo Fisher Scientific Co. Ltd., Waltham, USA). Fresh berries were smashed and being subjected to vacuum freeze-drying treatment. A mixed solvent of ethanol and ethyl acetate with the ration of 6:1 (v/v) was added to the powdered material to accumulate linalool and derivatives. The extract was then evaporated to dryness and the remaining mixture was resuspended in 200 μL of methanol before HPLC-HRMS analysis. Notably, some of the linalool derivatives accumulated in wine grape berries were glycosylated and further acid hydrolyzed under the pH 3 conditions, allowing for the complete quantification of these compounds (Ilc et al., 2017; Peng et al., 2024; Xia et al., 2024). Data were presented as the means ± SE (n = 3). Letters represent significant differences at P ≤ 0.05, as determined using ANOVA followed by Fisher's LSD test.

2.4. Isolation and sequence analysis of VvCYP76F14 and VvCPR1

The coding sequence (CDS) of VvCYP76F14 and VvCPR1 were cloned from wine grape cultivar ‘Heihuxiang’. Total RNA was extracted from the berries using a MiniBEST Plant RNA Extraction Kit (TaKaRa, Dalian, China), and any remaining DNA contamination was removed using RNase-free Recombinant DNase I (TaKaRa, Dalian, China). The RNA quantity and quality of the extracted RNA were assessed using an Invitrogen Qubit Flex Fluorometer (Thermo Fisher Scientific, Waltham, USA). Subsequently, the first-strand cDNA was synthesized using a PrimeScript II First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China).

The CDS of VvCYP76F14 was amplified using the Prime STAR™ HS DNA polymerase (TaKaRa, Dalian, China) with the specific primer pairs (Forward: 5′-ATGGAGTTGTTGAGTTGTCTG-3′; Reverse: 5′-TCAAACCCGTACAGGTAGAGCTT-3′) as previously described (Peng et al., 2024). The CDS of VvCPR1 was amplified using the Prime STAR™ HS DNA polymerase (TaKaRa, Dalian, China) with the specific primer pairs (Forward: 5′-ATGCAATCGAGCTCTGTGAAG-3′; Reverse: 5′-TCTAGAGATCCCTGCTCTTGC-3′) as previously described23. PCR products were then cloned into pMD 18-T (TaKaRa, Dalian, China) and sent for sequencing by Sangon Biotech Co., Ltd. (Shanghai, China). The resulting sequence was analyzed and translated into amino acid sequences using DNAMAN software. A phylogenetic tree was constructed using the Maximum-likelihood method in MEGA 13.0, including VvCYP76F14 proteins and CYP76 family homologues from 29 plant species.

2.5. Molecular docking analysis

Modeler v9.19 (http://salilab.org/modeller/) was utilized for homology modelling of the VvCYP76F14 protein from ‘Heihuxiang’, using the crystal structure of a Salvia miltiorrhiza CYP 450 protein (Li et al., 2021) (Protein Data Bank no. 5YLW) as the template. The protein underwent energy minimization treatment and served as the receptor structure for molecular docking. The stereochemical quality of the 3D model of VvCYP76F14 was evaluated using PROCHECK and Verify3D (Isin & Guengerich, 2007; Li et al., 2021).

The structures of the substrates (linalool, (E)-8-hydroxylinalool, and (E)-8-oxolinalool) were optimized using the MOPAC program and constructed using AutoDock4.2 (Morris et al., 2010). The Amber14 force field was utilized to perform energy optimization, ensuring the exclusion of any unreasonable spatial structures and stabilization of the binding models (Case et al., 2005). According to the description of molecular mechanics-generalized born surface area (MM-GBSA) method (Peng et al., 2024; Song et al., 2021; Xia et al., 2024), molecular dynamics simulations were conducted to estimate the relative binding free energy (ΔGbind) between the VvCYP76F14 and substrate (Supplementary Table 1). For each VvCYP76F14–ligand reaction system, the contributions of van der Waals (vdW), electrostatic interactions, and solvation (polar and nonpolar) were calculated to gain insights into the molecular conformations (Supplementary Table 1). The calculated energies of barriers can be expressed as:

k=kBTħexp−ΔG≠RT

where k, kB, T, ħ, −ΔG≠, and R represent the rate constant of reaction, Boltzmann's constant, the reaction temperature (setting at room temperature), Planck's constant, free energy difference between the reactant and the transition states, and the gas constant, respectively (Hannemann et al., 2007; Peters, 2017).

2.6. Site-directed mutagenesis (SM)

To reveal the catalytic activities of key amino acid residues in VvCYP76F14 from ‘Heihuxiang’, alanine-scanning method was employed to generate alanine-substituted VvCYP76F14-SMs (Peng et al., 2024; Xia et al., 2024; Song et al., 2021;). Triplet codons were optimized and synthesized at GenScript Co. Ltd. (Nanjing, China). Then, the VvCYP766F14-SMs were synthesized using the QuikChange Lightning Site Directed Mutagenesis Kit (Agilent Stratagene, New York, USA) and sequenced for validation by GenScript Co. Ltd. (Nanjing, China). All these key candidate amino acid residues were substituted with corresponding opposite polarity amino acids, respectively.

2.7. In vitro enzymatic activity assay

In order to efficiently express and facilitate the folding of recombinant VvCYP76F14s and VvCPR1 proteins, the pMAL-c6T vector (New England Biolabs, Beijing, China) containing a maltose-binding protein (MBP) tag was used for heterologous expression, as our previously described (Peng et al., 2024; Xia et al., 2024). The CDS of ‘Heihuxiang’ VvCYP76F14 and VvCYP760F14-SMs was cloned into the pMAL-c6T vector, respectively, to obtain the recombinant plasmids of pMAL-c6T-VvCYP76F14 or pMAL-c6T-VvCYP76F14-SMs. The recombinant plasmid was further validated by sequencing in Sangon Biotech Co., Ltd. (Shanghai, China), and then expressed in Escherichia coli BL21 strain (TaKaRa, Dalian, China). According to the manufacturer's description, VvCYP76F14-MBP or VvCYP76F14 MBP-SMs were further purified using the affinity between MBP and amylose resin by the NEBExpress MBP Fusion and Purification system (New England Biolabs, Hickin, UK). Qualitative and quantitative analyses of recombinant proteins was carried out by Shanghai Bioprofile Technology co., ltd (Shanghai, China).

In vitro enzyme activity levels of recombinant VvCYP76F14 and VvCYP76F14-SMs were independently assayed using linalool, (E)-8-hydroxylinalool, and (E)-8-oxolinalool as the substrate, respectively. Preliminary assays were conducted to determine the optimum reaction system. To reconstitute the membrane-bound monooxygenase system, the CPR homologue VvCPR1 (Hansen et al., 2021) present in wine grape itself was selected as the electron transport redox partner of VvCYP76F14. The VvCYP76F14 enzyme assays were performed in a total reaction volume of 5 mL with 100 mM Na+/K+ phosphate buffer (pH 5.0), varying substrate concentrations, 1 mM NADPH, and adjusted enzyme amounts (VvCYP76F14:VvCPR1 ≈ 2:1). The reactions were performed at 26 °C for 1 h with agitation, and the resulting products were collected and analyzed using HPLC-HRMS (Thermo Fisher Scientific Co. Ltd., Waltham, USA). Boiled protein (nonfunctional) was used as a control. All assays were performed in sextuplicate. For the determination of kinetic parameters, substrate reduction was qualitatively and quantitatively determined by HPLC-HRMS (Thermo Fisher Scientific Co. Ltd., Waltham, USA). The Kcat and Km were calculated using Origin 2020 online software (www.originlab.com).

2.8. Enzymatic treatment of wine

The enzymatic treatment of wine was conducted following the method of González-Pombo et al. (2014), with some modifications. Briefly, the above purified enzyme complex VvCYP76F14-VvCPR1 (1 ml) was added to 50 ml of the young wine at 25 °C for 50 min under agitation. Then, the wines were racked, filtered, bottled and stored for subsequent analysis. The wine bouquet compositions were analyzed weekly and compared with the excellent quality commercial wine, which had been aging (bottled storage) for 2 years. The content of linalool, (E)-8-hydroxylinalool, (E)-8-oxolinalool, (E)-8-carboxylinalool, and wine lactone in the wine grape berries were well glycosylated and determined HPLC-HRMS (Thermo Fisher Scientific Co. Ltd., Waltham, USA), and the acid hydrolysis of wine grape samples was carried out at pH 3. Six biological replicates were performed, each with 40 individual berries. A control experiment without the enzyme complex VvCYP76F14-VvCPR1 was conducted under the same conditions (Control wine).

2.9. Co-IP determination

The full-length VvCYP76F14 and VvCPR1 were cloned into the pBWA(V)Hs-TMVΩ-eGFP and pAN580–4 × MYC vectors, respectively. Both constructs were utilized to transform A. tumefaciens strain GV3101 (pSoup-P19, Weidi Biotechnology Co., Ltd., Shanghai, China) and were subsequently expressed in N. benthamiana leaves. The co-immunoprecipitation assays were conducted following the description of previous studies (Xu et al., 2020). Anti-GFP magnetic beads (Merck Co., Ltd., Darmstadt, Germany) were used to immunoprecipitate the protein complexes. The GFP and MYC signals were detected by anti-GFP and anti-MYC (Merck Co., Ltd., Darmstadt, Germany) antibodies.

2.10. Sensory analysis

Wine sensory analysis was carried out by a panel composed of 20 sommeliers following the guidelines outlined in GB/T 15037–2006 (January 2022) and ISO13301:2018 in three sessions. The visual characteristics, taste profiles, and aromatic attributes of the young wine and enzymatic treated wines were evaluated by the panels. The sensory experiments were performed using an interval scale from 0 to 10, with 0 indicating no smell of the aroma and 10 indicating a strong intensity of the aroma. Each panel member repeated the experiment three times, and the average value was taken as the fragrance intensity value. To further confirmed the correlation between concentration and sensory response, a sigmoid (S) curve was calculated based on a dose-response function:

y=A1+A2−A11+10LOGx0−xp

3. Results

3.1. Isolation VvCYP76F14 from high wine bouquet grape varieties

As a crucial precursor of wine lactones, (E)-8-carboxylinalool is directly implicated in the formation of wine bouquet (Alegre et al., 2020; Ilc et al., 2017; Noguerol-Pato et al., 2012). Based on the (E)-8-carboxylinalool content, 13, 11, and 7 wine grape varieties or superior lines were preliminarily categorized into Neutral, Aromatic, and Full-bodied types, respectively (Fig. 1). Notably, ‘Heihuxiang’ was found to possess a higher concentration of (E)-8-carboxylinalool (Fig. 1) and was consequently selected for coding sequence (CDS) cloning of VvCYP76F14 in this study.

Fig. 1.

Fig. 1

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Determination of (E)-8-carboxylinalool concentration in different wine grape varieties. All wine grape berries at the same berry maturity stage were selected for (E)-8-carboxylinalool determination, using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS, Waters, Milford, MA, USA), following the method previously described9. Data were presented as the means ± SE (n = 3). Letters represent significant differences at P ≤ 0.05, as determined using ANOVA followed by Fisher's LSD test.

Structural analysis of VvCYP76F14 in the grape genome revealed that one intron was observed in the genomic DNA sequence and the phase 0 intron position was located after the triple codon that encoding the amino acid residue of D299 (Fig. 2a). Both the AGTDT motif (monooxygenase P450) and the FGAGRRICFG motif (HEME-binding region) were present in VvCYP76F14 (Fig. 2b). According to the phylogenetic analysis, two conserved motifs were selected from plant CYP homologues, using Salvia miltiorrhiza CYP76 (which functions as a hydroxylase, Li et al., 2021) as the outgroup. Notably, the VvCYP76F14 protein exhibited a close relationship with the corresponding CYP76 homologue from Santalum spicatum (Fig. 2c).

Fig. 2.

Fig. 2

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Gene structure, amino acid sequence and phylogenetic tree analysis. (a) Gene structure of VvCYP76F14 cloned from wine grape cultivar ‘heihuxiang’. (b) Amino acid sequence analysis. Conserved regions are highlighted in red frames. (c) Phylogenetic analysis of plant CYP76F14 homologues. The phylogenetic tree of CYP76F14 homologues from wine grape and other 29 plant species, using the maximum-likelihood method in MEGA 13.0 software. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.2. Computer modelling and molecular docking analysis of VvCYP76F14

Among these VvCYP76F14–ligand reaction system, the van der Waals (ΔHMMvdWHMMvdW) component made the most significant contribution to the total free energy in each complex (Supplemental Table 1). In the dehydrogenation reaction system, the interaction energy of the VvCYP76F14–(E)-8-hydroxylinalool complex was found to be stable (−85.32 ± 3.19 kcal/mol). Computer modelling and molecular docking studies of VvCYP76F14 were conducted to predict the putative mechanism for the binding of linalool, (E)-8-hydroxylinalool, and (E)-8-oxolinalool with VvCYP76F14 during the trisubstrate-triproduct reaction (Fig. 3). Based on the stereochemical quality and verification analysis, the VvCYP76F14 model meets the evaluation criteria. The ‘semi-empirical free energy force field’ component of the molecular docking software was specifically chosen to analyze the involvement of amino acids in enzyme-substrate recognition. Through energy minimization, the algorithm identified the most reasonable binding conformations between VvCYP76F14 and the substrates (Homeyer & Gohlke, 2012). Subsequently, the substrates were docked to the active center of VvCYP76F14, forming a binding conformation pocket (Fig. 3a).

Fig. 3.

Fig. 3

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Computer modelling and molecular docking-based structure analysis revealed key amino acid residues involved in the monooxygenase reaction of VvCYP76F14. (a) Prediction of a binding conformation pocket in the active center of VvCYP76F14. (b) Prediction of key amino acid residues involved in the VvCYP76F14-linalool reaction. (c) Prediction of key amino acid residues involved in the CYP76F14–(E)-8-hydroxylinalool reaction. (d) Prediction of key amino acid residues involved in the CYP76F14–(E)-8-carboxylinalool reaction.

In the VvCYP76F14-linalool (hydroxylation) reaction, 17 amino acid residues (L91, R101, I120, R130, L298, V368, L371, R374, E378, T380, P434, F435, G436, A437, R440, I441, and C442) were predicted to form a reaction pocket during substrate binding to the VvCYP76F14 active site (Fig. 3b). In the subsequent VvCYP76F14-(E)-8-hydroxylinalool (dehydrogenation) complex, 19 amino acid residues (F138, R175, L185, T188, I189, V273, L297, D299, L300, F301, A302, L303, G304, P443, G444, L445, P446, A448, and M451) were found in the CYP76F14–(E)-8-hydroxylinalool complexes (Fig. 3c). Lastly, 18 amino acid residues (A302, L303, D306, T307, T311, S319, F362, H365, A367, V368, L372, P434, F435, C442, G483, I484, S485, and L486) were predicted to be involved in the CYP76F14-(E)-8-oxolinalool (carboxylation) reaction (Fig. 3d).

3.3. The physical association between VvCYP76F14 and VvCPR1 was confirmed by co-IP determination

To establish that VvCYP76F14 actually interacts with VvCPR1 in wine grape, Co-IP assays were performed in N. benthamiana leaves co-transformed with VvCYP76F14-GFP (VvCYP76F14 coupled with a GFP tag) and VvCPR1-MYC (VvCPR1-MYC coupled with a MYC tag). The Co-IP assay was divided into two groups: the input group and the IP group (Fig. 4a). The input group served as a positive control to determine whether the Co-IP system could detect the target proteins in the total protein from cell lysate. In the IP group, VvCYP76F14-GFP was used as the bait protein to form antigen–antibody complexes. The results showed that all the target proteins could be detected in the input group, confirming that the success of the entire protein expression system (Fig. 4a). In the IP group, the interaction between VvCYP76F14 and VvCPR1 was confirmed using the MYC-tag antibody but not with the GFP control (Fig. 4a). These findings implying that VvCYP76F14 interacts with VvCPR1 in wine grape.

Fig. 4.

Fig. 4

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Interaction verification between VvCYP76F14 and VvCPR1. (a) Co-IP verification. The plasmids of pBWA(V)Hs-TMVΩ-eGFP and pAN580–4 × MYC vectors transformed into A. tumefaciens strain GV3101and then were subsequently expressed in N. benthamiana leaves. Anti-GFP magnetic beads were used to immunoprecipitate the protein complexes. The GFP and MYC signals were detected by anti-GFP and anti-MYC antibodies. (b) Evaluation of the activity of VvCYP76F14 (Kcat/Km) with and without VvCPR1 in each reaction. Box plots were used to represent the data, including minimum, maximum, median and quartiles. Data are presented as means ± SE (n = 3). ** indicate significant differences at a significance level of P ≤ 0.05, as determined using a two-way ANOVA.

3.4. VvCPR1 is indispensable for the VvCYP76F14-linalool and VvCYP76F14-(E)-8-oxolinalool reactions in vitro

To better clarify the crucial role of VvCPR1 in each reaction, we conducted reaction systems with or without VvCPR1. The CDSs of VvCYP76F14 and VvCPR1 genes were heterologously expressed in the E. coli expression system. HPLC-HRMS data showed sequence coverage rates of 96.50 % and 94.30 % for the hydrolyzed peptide segments of heterologously expressed proteins in VvCYP76F14 and VvCPR1, respectively, confirming the identity of the recombinant enzymes from E. coli.

The enzyme activity was calculated using the turnover number (Kcat) to affinity (Km) ratio. In the VvCYP76F14-linalool (hydroxylation) and VvCYP76F14-(E)-8-oxolinalool (carboxylation) reaction systems, the enzyme catalysis could not proceed without VvCPR1 (Fig. 4b). However, in the VvCYP76F14-(E)-8-hydroxylinalool (dehydrogenation) reaction system, enzyme catalysis occurred normally with and without VvCPR1, and there was no significant difference between the two reactions (Fig. 4b). These findings suggest that NADPH-cytochrome P450 reductase is essential for the hydroxylation and carboxylation processes of VvCYP76F14 but is not necessary for the dehydrogenation process.

3.5. D299 was identified as the electron transport redox partner in the dehydrogenation reaction process, instead of VvCPR1

To further understand the mechanism behind why VvCPR1 is indispensable for the VvCYP76F14-linalool and VvCYP76F14-(E)-8-oxolinalool reactions but not for the VvCYP76F14-(E)-8-hydroxylinalool (dehydrogenation) reaction, we conducted substitutions of candidate key amino acid residues based on the prediction of computer modelling and molecular docking (Fig. 3), and examined each candidate amino acid residue's role in VvCYP76F14's reactions using site-directed mutagenesis (SM).

In the VvCYP76F14-linalool (hydroxylation) reaction, independent substitutions of all 17 candidate amino acid residues, except for V368, L371, and P434, may decrease the enzyme's efficiency (Kcat/Km ratio) (Fig. 5a). In the VvCYP76F14-(E)-8-hydroxylinalool (dehydrogenation) reaction, independent substitution of 16 out of 19 candidate amino acid residues produced significant effects on the enzyme's efficiency (Kcat/Km ratios) (Fig. 5b). Notably, the substitution of D299 led to a complete loss of enzyme activity. In the CYP76F14-(E)-8-oxolinalool (carboxylation) reaction, independent substitution of each candidate amino acid residue, except for I484, decreased the enzyme activities of the corresponding CYP76F14-SMs (Fig. 5c).

Fig. 5.

Fig. 5

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Enzyme kinetics of VvCYP76F14 and its site-directed mutant proteins (VvCYP76F14-SMs) using linalool, (E)-8-hydroxylinalool, and (E)-8-oxolinalool as substrate, respectively. Data were presented as the means ± SE (n = 3). Letters indicate significant differences at a significance level of P ≤ 0.05, as determined using ANOVA followed by Fisher's LSD test. N.D., not detectable.

Furthermore, we estimated the energy barriers related to the reaction rates without the electron transport redox partner. Considering only the ‘effective’ candidate amino acid residues, the barriers were 49.74 (hydroxylation), 17.16 (dehydrogenation), and 52.27 (carboxylation) kcal/mol, respectively. According to the Transition State Theory (TST) (Peters, 2017; Tantillo, 2021), only the dehydrogenation reaction was feasible, while the hydroxylation and carboxylation reactions were not (usually, the enzymatic reaction pathway is considered unfeasible when the barrier is above 20 kcal/mol). Interestingly, the energy barrier of the dehydrogenation reaction was calculated as 36.29 kcal/mol when D299 was removed from the list of candidate amino acid residues. These findings suggest that D299, rather than NADPH-cytochrome P450 reductase, might be used as the electron transport redox partner during the process of dehydrogenation reaction.

3.6. Influence of VvCYP76F14-VvCPR1 complex treatment on wine lactone composition

To evaluate the effects of enzymatic treatment on enhancing the potential wine bouquet, the VvCYP76F14-VvCPR1 complex was added to the young wines (derived from ‘Heihuxiang’). The HPLC-HRMS results showed that the precursors of wine lactone (linalool, (E)-8-hydroxylinalool, (E)-8-oxolinalool, and (E)-8-carboxyllinalool) were significantly increased in enzymatic treatment samples (Fig. 6a). There were low levels of (E)-8-hydroxylinalool, (E)-8-oxolinalool, and (E)-8-carboxyllinalool in the control sample wines.

Fig. 6.

Fig. 6

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The impact of VvCYP76F14-VvCPR1 complex treatment on wine bouquet composition. (a) The precursors of wine lactone were obviously increased in enzymatic treatment samples after 5-weeks aging and the wines with 2 years aging, whereas undetected in the control wines. (b) The content of wine lactone and the precursors. (c) The effects of VvCYP76F14-VvCPR1 complex treatment on wine bouquet intensity.

Wine lactone also significantly increased in those enzymatic treatment samples and was undetectable in the control samples (Fig. 6b). Notably, after 5-weeks of “aging”, some of the wines with enzymatic treatment achieved similar quantities of bouquet compositions (wine lactone and its precursors) as the commercial wines aged for 2 years, which are considered to have the best bouquet quality by sommelier panels (Fig. 6b). Next, we were interested in determining whether VvCYP76F14-VvCPR1 complex treatment correlated with the improvement of wine bouquet in other Neutral and Aromatic wines. Therefore, we selected 16 Neutral and Aromatic type wines for bouquet composition analysis. The concentrations of wine lactone and its precursors in the enzymatic treatment samples were significantly superior to those in the control wines.

3.7. Enzymatic treatment influence on sensory data

To evaluate the effect of VvCYP76F14-VvCPR1 complex treatment on wine bouquet, a descriptive sensory analysis of wine bouquet intensity was performed. According to the sommelier panel, the sensory analysis was consistent with the above-obtained results, showing that 5-week-aged wine samples, which obtained similar bouquet compositions to the commercial wines aged for 2 years, also exhibited similar bouquet intensity to the commercial wines. To further confirm the potential of the enzyme complex for bouquet development in wines, the wine lactone in ‘5-weeks aging’ samples and the 2-years aging wine were diluted to the same concentration. The data from the S-curve, which showed whether the 5-weeks aging enzymatic treatment samples (R2 = 0.992) or commercial wine (R2 = 0.996) significantly increased the intensity of the wine bouquet as the concentration of wine lactone increased (Fig. 6c), indicating that the VvCYP76F14-VvCPR1 complex has the potential to shorten the aging time.

4. Discussion

In wine grapes, the enzyme VvCYP76F14 is involved in three reaction processes (hydroxylation, dehydrogenation, and carboxylation) using linalool as a substrate (Ilc et al., 2017). However, the detailed mechanism of the complete enzymatic cycle for this multi-substrate enzyme and the specific amino acid residues contributing to wine bouquet formation remains largely unknown.

In eukaryotes, Class II cytochrome P450s in the endoplasmic reticulum employ NADPH-cytochrome P450 reductase (CPR) as an electron donor during the hydroxylation reaction process (Jensen & Møller, 2010; Peng et al., 2024; Zhang et al., 2020). In this study, the involvement of a redox partner was found to be essential for the VvCYP76F14-linalool (hydroxylation) and VvCYP76F14-(E)-8-oxolinalool (carboxylation) reactions, but possibly not for the VvCYP76F14-(E)-8-hydroxylinalool (dehydrogenation) reaction. Specifically, the hydroxylation and carboxylation reactions catalyzed by VvCYP76F14 involve the introduction of an O-atom, resulting in the insertion of a hydroxyl group into the linalool or (E)-8-oxolinalool substrates. These two oxygenation processes are characteristic chemical reactions of cytochrome P450 monooxygenase in the endoplasmic reticulum of plant cells (Hannemann et al., 2007; Isin & Guengerich, 2007; Jensen & Møller, 2010). Here, the necessary proton-electrons pairs for catalysis are provided by CPR (Fig. 7). On the other hand, the reaction involving VvCYP76F14-(E)-8-hydroxylinalool is a typical dehydrogenation process that might not require VvCPR1 for electron transfer. Based on these observations, we can propose a hypothesis: In trisubstrate-triproduct reactions, only the oxygenation processes involving the introduction of an exogenous O-atom require the participation of VvCPR1 in the proton-electron transport chain (Fig. 7a), while the dehydrogenation oxidation process likely does not (Fig. 7b). This hypothesis can also explain why site-directed mutagenesis of the candidate amino acid residues in the VvCYP76F14-linalool and VvCYP76F14-(E)-8-oxolinalool reactions did not lead to complete inactivation of the enzyme, whereas mutation of the key site D299 in the VvCYP76F14-(E)-8-hydroxylinalool reaction resulted in the complete loss of the enzyme activity. In the dehydrogenation reaction, D299 is involved in the proton-electron transfer chain instead of VvCPR1 (Fig. 7c). It is noted that aspartic acid (Asp, D) residues in various natural enzymes are prone to play a role in proton transfer during redox reactions (Ribeiro et al., 2020). Similar findings have been observed in peach, where the D376 mutation in PpAAT1 led to the complete loss of γ-decalactone biosynthesis activity in the internal-esterification reaction (Song et al., 2021). Notably, D299 was located at the intron positions of VvCYP76F14 (Fig. 2a). Intron positions and phases can serve as distinguishing characteristics for classifying functionally differentiated proteins (Xue et al., 2012). To the best of our knowledge, plant NADPH-cytochrome P450 oxidoreductases that require CPR are hydroxylase (Hildreth et al., 2020; Jensen & Møller, 2010; Paine et al., 2005; Werck-Reichhart & Feyereisen, 2000). In this study, these two steps of reaction (hydroxylation and carboxylation) were fundamentally hydroxylation processes. This could be one of the reasons why these two reactions require VvCPR1. We further surveyed NADPH-cytochrome P450 oxidoreductase genes in various angiosperms, focusing on intron positions and phases (specifically, intron phase at key amino acid residues), to explore whether this phenomenon contributed to the evolution of multi-substrate-product (trisubstrate-triproduct) functions.

Fig. 7.

Fig. 7

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Proposed catalytic mechanism of VvCYP76F14 in the hydroxylation, dehydrogenation and carboxylation reactions. (a) VvCPR1 was found to be essential for hydroxylation and carboxylation using Linalool and (E)-8-oxolinalool as substrates, respectively. (b) VvCPR1 might not be involved in dehydrogenation using (E)-8-hydroxylinalool as substrate. (c) The proposed catalytic mechanism of VvCYP76F14 in the dehydrogenation process. Notably, Asp 299 is interacted with the hydroxyl group of (E)-8-hydroxylinalool. The hydrogen atom of the hydroxyl group of (E)-8-hydroxylinalool is transferred to the carboxyl group of Asp299, resulting in the formation of a positively charged hydroxyl group and carbon‑oxygen double bond in (E)-8-hydroxylinalool. Ultimately, (E)-8-oxolinalool is formed. (d) Schematic of the complete enzymatic cycle of VvCYP76F14 in trisubstrate-triproduct reactions. ①, ②, and ③ indicates the hydroxylation, dehydrogenation, and carboxylation reaction process, respectively.

It is widely acknowledged that the overall enzymatic rate in many natural enzymes is often limited by the rate at which products are released, known as the rate-limiting step (Benkovic & Hammes-Schiffer, 2003; Cleland, 1975). Natural multi-substrate enzymes have evolved to facilitate this rate-limiting step in the enzymatic cycle, often involving conformational changes of the enzyme. An example is the bisubstrate-biproduct adenylate kinase, where conformational motions facilitate the product-release process (Karplus, 2010; Saavedra et al., 2018). Recently, the Steric Frustration model has been developed to explain how bisubstrate-biproduct enzymes facilitate the product-release process throughout the entire enzymatic cycle (Li et al., 2021). Applying this model, it is reasonable to propose that the trisubstrate-triproduct enzyme VvCYP76F14 has evolved to catalyze reactions with high efficiency by coordinating the substrate-binding and product-release steps in the complete enzymatic cycle (Fig. 7d). Initially, linalool binds to its specific binding site and is hydroxylated to (E)-8-hydroxylinalool by the VvCYP76F14-VvCPR1 system. Considering that the product of the previous reaction may serve as the substrate for the next reaction, substrate-product exchange occurs through conformational motions, resulting in the separation of VvCYP76F14 from VvCPR1. The (E)-8-hydroxylinalool is then dehydrogenation-oxidized to (E)-8-oxolinalool at the next site by VvCYP76F14. Subsequently, (E)-8-oxolinalool is carboxylated by the VvCYP76F14-VvCPR1 system. During these steps, the binding of the chemicals at the sites leads to the formation of a substrate-product co-bound complex, resulting in steric incompatibility. As a result, the bottleneck products are squeezed out due to the steric frustration of the CPR enzyme system. Further crystallography studies of VvCYP76F14 would be necessary to confirm and identify this proposed model.

In recent years, the improvement of aroma compounds and their precursors has been placed on glycosidases during the vinification process (González-Pombo et al., 2014; Sun et al., 2018). These enzyme treatment strategies could increase some monoterpenes and enrich some wine aromas, such as esters and phenylethyls. However, these commercial hydrolases might not increase the derivatives of linalool and thus fail to improve the wine bouquet. For example, seven different commercial glycosidases did not significantly contribute to the enhancement of linalool contents and the wine bouquet (Rusjan et al., 2012). Although the initial composition and content of monoterpenes of the commercial wine may differ from that of the enzyme-treated wines, the VvCYP76F14-VvCPR1 complex significantly improved the bouquet by increasing the content of wine lactone and shortening the aging time when the VvCYP76F14-VvCPR1 complex was introduced in the aging stage, and the sensory evaluation confirmed these results. Moreover, the effects of VvCYP76F14-VvCPR1 complex treatment on wine bouquet intensity were further confirmed by the S-curve analysis (Fig. 6c). For the changes within 5 weeks, it is necessary to focus on one sample to be more accurate that we will continue to study in the future. Nonetheless, this study indicate that the VvCYP76F14/VvCPR1 enzyme system could be used to rapidly convert linalool into short-aging wine lactone.

5. Conclusions

VvCYP76F14 has likely evolved a Steric Frustration strategy to efficiently catalyze trisubstrate-triproduct reactions by facilitating the product-release steps in the complete enzymatic cycle. The CYP76F14-(E)-8-hydroxylinalool reaction is the rate-limiting step and VvCPR1 was indispensable for hydroxylation and carboxylation. The VvCYP76F14-VvCPR1 complex could improve the bouquet by increasing the content of wine lactone and shortening the aging time.

Ethics

Ethical permission was not required.

Consent

The panelists gave their consent to participate within sensory research.

CRediT authorship contribution statement

Zhizhong Song: Writing – review & editing, Project administration, Investigation, Funding acquisition, Conceptualization. Meiling Tang: Writing – review & editing, Resources, Investigation, Funding acquisition. Huilin Xiao: Writing – review & editing, Visualization, Formal analysis. Houhua Xu: Validation, Software, Investigation, Data curation. Matthew Shi: Methodology, Investigation, Formal analysis, Data curation. Adeeba Dark: Writing – original draft, Visualization, Formal analysis. Zhenqiang Xie: Writing – review & editing, Investigation, Data curation. Bin Peng: Writing – review & editing, Project administration, Investigation, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors are grateful to Professor Julia M. Davies, Department of Plant Sciences, University of Cambridge for critical reading. The authors are grateful to Dr. Jianping Qi, Thermo Fisher Scientific, for qualitative and quantitative determination of compounds in this study. This study was supported by grants from the Major Project of Science and Technology of Shandong Province (2022CXGC010605), Science and Technology Project of the Fourth Division of Xinjiang Production and Construction Corps (2024GG018), Science Fund of Jiangsu Vocational College of Agriculture and Forestry (2021kj23), Agriculture Research System of China of MOF and MARA (CARS-29-17), UKRI BBSRC (X008843/1), and Fund of China Scholarship Council (202208370080).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2025.143077.

Contributor Information

Zhenqiang Xie, Email: xiezhenqiang@jsafc.edu.cn.

Bin Peng, Email: pengbin@jsafc.edu.cn.

Appendix A. Supplementary data

Supplementary material: Supplementary Table 1 MM-GBSA analysis of VvCYP76F14-ligand complexes (kcal/mol).

mmc1.docx (16.9KB, docx)

Data availability

Data are available within the article or its supplementary materials.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material: Supplementary Table 1 MM-GBSA analysis of VvCYP76F14-ligand complexes (kcal/mol).

mmc1.docx (16.9KB, docx)

Data Availability Statement

Data are available within the article or its supplementary materials.

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