Elucidating the biochemical properties of limonene synthase from Agastache rugosa

Abstract

Background

Limonene, a monocyclic monoterpene abundant in citrus essential oils, is extensively utilized in the fragrance, flavor, and pharmaceutical industries. Limonene synthase (LS) is a key enzyme in limonene biosynthesis by converting geranyl pyrophosphate to limonene. Despite its industrial significance, microbial limonene production is limited to a few LSs due to expression challenges. There is a growing demand to discover and characterize new LS enzymes with enhanced catalytic efficiency and diverse functional properties. Although LS from Mentha spicata (MsLS) is commonly used for microbial limonene production, its industrial applicability is hindered by relatively low catalytic efficiency. Therefore, this study focuses on the characterization of LS from Agastache rugosa (ArLS), which has shown higher catalytic efficiency than MsLS under the tested conditions and may provide a valuable biochemical foundation for microbial limonene production. This study provides a comprehensive kinetic analysis and identifies putative active-site residues of ArLS using homology modeling and site-directed mutagenesis, techniques rarely applied to LSs.

Results

ArLS was cloned and heterologously expressed in Escherichia coli, leading to high limonene production, approximately 60% of the theoretical maximum based on the provided substrate, which was 1.15 times higher than that achieved with MsLS. The enzyme exhibited optimal activity at 30 °C and pH 8.0 in the presence of 0.1 mM Mn²⁺, aligning with the typical catalytic conditions of plant terpene synthases. Kinetic analysis indicated that ArLS displayed the highest catalytic efficiency among reported LSs, with a significantly lower K_M (5.5 ± 0.8 µM) and a higher k_cat/K_M (0.4 ± 0.06 min⁻¹·µM⁻¹). Structural insights into the putative active site of ArLS were obtained through homology modeling using AlphaFold. To validate these predictions, site-directed alanine scanning mutagenesis was performed on the putative active-site residues. Comparative analysis with previously characterized LSs revealed higher substrate-to-product conversion rate.

Conclusions

Characterization of ArLS highlights its potential for biotechnological limonene production. Its high catalytic efficiency and substrate affinity indicate that it is a promising candidate for industrial applications. This study offers the initial comprehensive kinetic characterization and putative active-site analysis of ArLS, enhancing our understanding of its catalytic mechanism. Enzyme assays, kinetic assessments, and structural insights from this study could establish a basis for future enzyme engineering to improve enzyme performance. Additionally, this study lays the groundwork for further investigation of monoterpene synthases and the sustainable production of valuable terpenes.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12870-026-09463-0.

Background

Terpenes are a diverse class of natural plant products that contribute to defense, ecological interactions, and various physiological processes, such as induced resistance, stress adaptation, and defense against insects and other herbivores [1–4]. These metabolites are synthesized by assembling isoprene units following the general formula (C₅H₈)_n (n ≥ 2), forming the largest category of natural products, with over 50,000 compounds identified to date [5–8]. Terpenes also possess significant industrial value, with established applications in food flavoring, cosmetics, pharmaceuticals, and emerging biofuel technologies [9]. The increasing focus on sustainable and cost-effective biotechnological strategies is transforming the production of terpenes. Traditional methods such as plant biomass extraction and chemical synthesis are hindered by high costs, low yields, and susceptibility to contamination [10, 11]. To overcome these challenges, metabolic engineering of microbial production has emerged as a promising strategy for the sustainable and cost-effective biosynthesis of high-value terpenes [12].

Among the terpenes, monoterpenes (C₁₀H₁₆), composed of two isoprene units, have garnered significant attention due to their diverse biological activities and potential as renewable and sustainable resources [13]. Limonene, a monoterpene of considerable industrial importance, exists in two enantiomers: (S)-limonene, present in coniferous trees as well as caraway, dill, and bergamot, emits a sharp, turpentine-like odor [14], while (R)-limonene, abundant in citrus plants and fruits [15, 16], exudes a sweet, orange-like fragrance, making it highly valued in consumer products [17]. Beyond its aromatic qualities, limonene demonstrates anti-inflammatory, antioxidant, and anticancer activities, further underscoring its pharmaceutical potential [18–21]. Additionally, limonene has emerged as a promising biofuel alternative as the hydrogenated monoterpenes derived from it enhance the cold weather performance of jet fuel mixtures [22]. Consequently, the development of efficient biotechnological platforms for limonene biosynthesis has become a research priority [23].

Limonene biosynthesis commences with geranyl pyrophosphate (GPP), a crucial precursor formed by the condensation of two isoprene units, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), sourced from the methylerythritol-4-phosphate (MEP) pathway or mevalonate (MVA) pathway [24]. Limonene synthase (LS) then catalyzes the cyclization of GPP into limonene through a series of carbocation-mediated rearrangements (Fig. 1) [25, 26]. While significant progress in synthetic biology has facilitated gram-scale monoterpene production in Escherichia coli and Saccharomyces cerevisiae [27–33], the expression of plant-derived LSs in these microbial hosts remains challenging owing to their structural complexity and limited solubility [34]. However, various LSs such as those from Mentha spicata (MsLS) [35], Lavandula angustifolia (LaLS) [36], Schizonepeta tenuifolia (StLS) [37], Cannabis sativa (CsLS) [38], Perilla frutescens (PfLS) [39], Citrus unshiu (CuLS) [40], Citrus limon (ClLS) [41], Abies grandis (AgLS) [42], and Picea abies (PaLS) [43], have been successfully expressed in E. coli. Despite these advances, most studies have been limited to phylogenetic analyses, gene identification, or basic functional characterization, with limited success in achieving highly efficient enzymatic conversion of GPP to limonene. Currently, (S)-LS from MsLS is predominantly used for microbial limonene production; however, its relatively low catalytic efficiency hinders its industrial application. Considering the high limonene content of LS from Agastache rugosa (ArLS) emerges as a promising alternative [44]. Although ArLS has been previously characterized, potential residues responsible for its stereoselectivity have been suggested [45], and recent research has highlighted substrate-specific behavior in E. coli production systems [46]. However, detailed biochemical characterization of the purified enzyme, encompassing kinetic parameters and experimental validation of structure–function relationships, remains limited.

Fig. 1.

Enzymatic cyclization mechanism of LS converting geranyl pyrophosphate into limonene

Therefore, this study aimed to provide a comprehensive functional and biochemical characterization of ArLS heterologously expressed in E. coli and purified. Reaction conditions such as pH, temperature, and metal ion concentration were optimized to enhance the enzymatic reaction. Furthermore, we conducted the first detailed kinetic analysis of ArLS, demonstrating a significantly higher catalytic efficiency and substrate affinity than those of previously reported LSs. This study further examined the ArLS active site using homology modeling and site-directed mutagenesis, thereby providing functional validation of the conserved residues involved in substrate stabilization and catalysis. These findings clarify the catalytic features of ArLS and provide fundamental insights to guide future enzyme engineering and industrial bioproduction of limonene.

Methods

Gene synthesis, cloning, and site-directed mutagenesis

ArLS (GenBank accession No. AAL17636) and MsLS (GenBank accession No. AAC37366) genes were synthesized by Cosmo Genetech (Seoul, Republic of Korea) in truncated form, excluding the predicted N-terminal plastid-targeting peptides (residues 1–68 and 1–56 for ArLS and MsLS, respectively) based on SignalP 6.0 predictions from DTU Health Tech (Lyngby, Denmark). No plant materials, seeds, or field-collected samples were utilized. The synthesized DNA fragments and the pET-28a(+) vector were amplified via polymerase chain reaction (PCR) using PrimeSTAR Max DNA Polymerase (Takara, Kusatsu, Japan) and the primers listed in Table S1. Subsequently, the amplified DNA fragments and linearized pET-28a(+) vector were ligated using Gibson Assembly Master Mix (New England BioLabs, Ipswich, USA), and the resulting recombinant plasmids were transformed into E. coli C2566. The transformed cells were cultured on Luria–Bertani (LB) agar containing 30 µg mL^–1 kanamycin. Following this, colonies were cultured, and plasmids were isolated using a QIAGEN plasmid purification kit (QIAGEN, Hilden, Germany). Site-directed mutagenesis of ArLS was performed to generate variants using the overlapping PCR method [47]. Single-point mutations in ArLS were induced by PCR utilizing the pET-28a(+)-ArLS plasmid as a template, with the primers listed in Table S1. The resulting ArLS variants were transformed into E. coli C2566 and purified as previously described above. Macrogen (Daejeon, Republic of Korea) conducted complete sequencing of all LS genes in the plasmid constructs.

Enzyme expression and purification

Plasmids encoding MsLS, ArLS, and their variants were transformed into E. coli C2566. Transformants were cultured in LB medium containing 30 µg mL^–1 kanamycin at 37 °C with shaking at 200 rpm. Upon reaching an optical density at 600 nm (OD₆₀₀) of 0.6, protein expression was induced with 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cultures were then incubated at 20 °C with agitation at 150 rpm for 16–20 h. Cells were harvested by centrifugation at 3,500 × g and 4 °C for 20 min. Recombinant E. coli cells were resuspended in ice-cold lysis buffer (20 mM Tris–HCl, 500 mM NaCl, pH 8.0) and disrupted on ice for 5 min through ultrasonication (Fisher Scientific, Pittsburgh, PA). Unbroken cells and debris were removed by centrifugation at 18,000 × g and 4 °C for 20 min. The supernatant was filtered through a 0.2 μm filter and then loaded onto a Ni²⁺-charged immobilized metal ion affinity chromatography column (BioWorks, Uppsala, Sweden) equilibrated with lysis buffer. The column was washed at least twice with wash buffer (20 mM Tris–HCl, 500 mM NaCl, and 10 mM or 20 mM imidazole, pH 8.0), and the bound protein was eluted with elution buffer (20 mM Tris–HCl, 500 mM NaCl, and 250 mM imidazole, pH 8.0). The eluted proteins were desalted using 30 kDa molecular weight cutoff ultracentrifugal filtration tubes (Amicon, Sigma-Aldrich, St. Louis, MO, USA) and dialyzed against 50 mM N-(2-hydroxyethyl)piperazine-Nʹ-(3-propanesulfonic acid) (EPPS, pH 8.0) at 4 °C. The concentration of the purified enzyme was determined using a Bradford assay with bovine serum albumin as the standard [48].

Molecular weight determination of limonene synthase from Agastache rugosa

The purified enzyme was analyzed under denaturing conditions using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with protein molecular weight markers as references. The subunit molecular mass of the purified ArLS was determined through gel filtration chromatography on a HiPrep 16/60 Sephacryl S-400 column (Cytiva, Amersham, England). The enzyme was loaded onto the column and eluted with 50 mM sodium phosphate buffer (pH 7.2) containing 150 mM NaCl at a flow rate of 0.5 mL min⁻¹. The column was calibrated using thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), carbonic anhydrase (29 kDa), and ribonuclease A (13.7 kDa) as reference proteins (Cytiva). The native molecular mass of the enzyme was determined based on the migration of the reference proteins.

Effect of temperature, pH, and metal ions on limonene synthesis

Unless stated otherwise, the reactions were performed with 0.1 mg mL⁻¹ enzyme, 50 µM GPP, and 5 mM Mg²⁺ at 25 °C for 15 min. To evaluate effects of pH on enzymatic activity of ArLS, reactions were conducted in 50 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES, pH 6.5–7.5), 50 mM EPPS (pH 7.5–8.5), or 50 mM 2-(cyclohexylamino)ethanesulfonic acid (CHES, pH 8.5–10.0). Temperature effects were evaluated in 50 mM EPPS (pH 8.0) buffer at 10, 20, 30, 40, and 50 °C. To evaluate the effects of metal ions, the purified enzyme was pretreated with 10 mM ethylenediaminetetraacetic acid (EDTA) for 1 h. The enzyme was then dialyzed against 50 mM EPPS (pH 8.0), and incubated overnight at 4 °C with 1 mM of each metal ion: Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Ca²⁺, and Zn²⁺. The treated enzyme samples were then used for the assay. The optimal metal concentrations were determined using Mg²⁺ (0–10 mM) and Mn²⁺ (0–5 mM). All experiments were performed in duplicate, and the data are presented as the mean of two independent measurements.

Specific activity and kinetic parameters

One unit (U) of enzyme activity was defined as the amount of enzyme that produced 1 nmol of limonene per minute. The kinetic parameters of ArLS and MsLS were determined using various concentrations of GPP as substrates. Reactions for ArLS were conducted in 50 mM EPPS buffer (pH 8.0) containing 0.01 mg mL^–1 of enzyme and 0.1 mM Mn²⁺ at 30 °C for 15 min. MsLS and limonene synthase from Mentha × piperita (a hybrid of Mentha spicata and Mentha aquatica) share 93% sequence identity. Based on the optimized conditions for LS extracted from Mentha × piperita [49],the enzymatic reactions for MsLS were performed under the previously reported reaction conditions: 50 mM PIPES buffer (pH 6.7), 0.005 mg mL⁻¹ enzyme, and 1 mM Mn²⁺ at 30 °C for 15 min. Kinetic parameters were calculated using the Michaelis–Menten equation with GPP concentrations ranging from 1 to 200 µM.

Limonene production from geranyl pyrophosphate by limonene synthase

To compare the limonene production capacity of ArLS and MsLS, enzyme concentrations were set at 5 mg mL⁻¹, and substrate concentrations were set at 1, 4, 7, and 10 µM. ArLS reactions were conducted in 50 mM EPPS buffer (pH 8.0) with 0.1 mM Mn²⁺ at 30 °C for 15 min, while MsLS reactions were conducted in 50 mM PIPES buffer (pH 6.7) with 1 mM Mn²⁺ at 30 °C for 15 min. Statistical analyses were performed using a two-tailed Welch’s t-test to compare ArLS and MsLS at each substrate concentration. Differences with p < 0.05 were deemed statistically significant and are denoted by an asterisk (*).

In silico analysis of limonene synthase from Agastache rugosa

To identify the mutant residues’ positions in the active site, we conducted de novo structure prediction of ArLS utilizing the AlphaFold2 framework (AlphaFold DB, https://alphafold.ebi.ac.uk) [50] and visualized the results with PyMOL v3.0 (Schrödinger, USA). The structure prediction process followed the guidelines provided on the AlphaFold web server [50]. Subsequently, the amino acid residues surrounding the ligand within the protein were identified and analyzed using BIOVIA Discovery Studio Visualizer (Dassault Systèmes, Vélizy-Villacoublay, France).

Sample preparation and gas chromatography analysis

An equal volume of hexane (1:1, v/v) was added to the reaction mixture during the enzymatic assay to extract limonene. Following incubation, the reactions were quenched by brief vortexing for 10 s, then vigorous vortexing for 5 min, and subsequent centrifugation at 13,000 × g for 10 min. The resulting hexane layer was collected and analyzed using gas chromatography–flame ionization detection (GC–FID). Limonene standards (1–50 µM) were prepared in 50 mM EPPS (pH 8.0) containing 1% Dimethyl Sulfoxide (DMSO), with sec-butylbenzene (64 µM) serving as an internal standard for quantification (Fig. S1).

Samples were analyzed via GC–FID using a GC-2030 gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector and an HP-5MS column (30 m × 0.25 mm × 0.25µm; Agilent, USA). The injector and detector temperatures were set to 250 °C and 300 °C, respectively. Each sample (1 µL) was injected in split mode (2:1). The oven temperature was initially sustained at 50 °C for 1 min, increased to 190 °C at 8 °C min⁻¹, and then to 300 °C at 40 °C min⁻¹ for 5 min. Helium was used as the carrier gas at a flow rate of 1.2 mL min⁻¹.

Results

Identification and expression of limonene synthase from Agastache rugosa

The ArLS gene was cloned and analyzed at the nucleotide and protein levels to characterize its molecular features. Amino acid sequence alignment using SnapGene 5.2.4 (SnapGene, San Diego, USA) revealed that ArLS shared 71.45% identity with MsLS, a well-characterized limonene synthase. Consistent with other plant terpene synthases, ArLS contained the conserved metal-binding motifs Asp-Asp-Xxx-Xxx-Asp (DDXXD) and (Asn/Asp)-Asp-(Leu/Ile/Val)-Xxx-(Ser/Thr)-Xxx-Xxx-Xxx-Glu (NSE/DTE), crucial for coordinating divalent metal ions during catalysis (Fig. 2a).

Fig. 2.

Identification and characterization of ArLS. a Amino acid sequence alignment of ArLS with MsLS. The signal peptide region boxed in yellow rectangle and AlphaFold-predicted secondary structures indicated by red wavy lines. Conserved metal-binding motifs (DDXXD and NSE/DTE) boxed in blue. Conserved residues shaded in sky blue, and asterisks above the sequence indicate catalytic residues forming the substrate-binding pocket. b SDS–PAGE analysis of recombinant MsLS and ArLS. Crude extracts and purified enzymes of each LS were loaded alongside the middle lane, protein markers (130, 95, 70, 56, 43, 35, and 28 kDa, from top to bottom). c Determination of the native molecular mass of ArLS via GPC. Reference proteins (black circles) include ribonuclease A (R, 13.7 kDa), carbonic anhydrase (CA, 29 kDa), conalbumin (C, 75 kDa), aldolase (Ald, 158 kDa), ferritin (F, 440 kDa), and thyroglobulin (T, 669 kDa). The molecular mass of ArLS is indicated using a red star

For functional expression in E. coli, the plastid-targeting peptides of ArLS and MsLS were eliminated. The resulting recombinant enzymes were efficiently expressed, and SDS–PAGE analysis confirmed that ArLS and MsLS exhibited the expected molecular masses of 66.1 kDa and 66.5 kDa, respectively (Fig. 2b). These values were consistent with the calculated sizes of their mature forms. To assess the oligomeric state of ArLS, the native enzyme underwent analysis via gel permeation chromatography (GPC). The elution profile indicated an estimated molecular mass of approximately 137 kDa, suggesting that ArLS primarily exists as a dimer in solution (Fig. 2c). These results collectively indicate that ArLS possesses conserved sequence features typical of terpene synthases, is effectively expressed in E. coli, and forms dimeric structures under native conditions.

Biochemical properties of limonene synthase from Agastache rugosa

Optimal reaction conditions, such as temperature, pH, and metal ions, are crucial for achieving maximum catalytic efficiency. The biochemical properties of ArLS were characterized by measuring its capacity to convert GPP into limonene using GC–FID analysis. ArLS activity peaked at pH 8.0 and decreased to below 65% at other pH values (Fig. 3a). Maximum activity was observed at 30 °C, with a significant decrease at higher temperatures (Fig. 3b). Since LSs depend on divalent metal ions for catalysis [51], the effects of Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Ca²⁺, and Zn²⁺ on ArLS activity were investigated. ArLS maintained nearly 100% activity with Mg²⁺ or Mn²⁺, while other ions did not facilitate catalysis (Fig. 3c). Examination of various concentrations of Mg²⁺ and Mn²⁺ revealed that 0.1 mM Mn²⁺ yielded optimal activity, whereas higher concentrations led to inhibition (Fig. 3d). In summary, the ideal conditions for ArLS-mediated limonene biosynthesis were pH 8.0, 30 °C, and 0.1 mM Mn²⁺.

Fig. 3.

Effects of pH, temperature, and metal ions on ArLS activity. a Effect of pH on ArLS activity. Reactions were conducted in PIPES (pH 6.5–7.5), EPPS (pH 7.5–8.5), or CHES (pH 8.5–10.0) containing 0.1 mg mL⁻¹ enzyme, 50 μM GPP, and 5 mM Mg²⁺ at 30 °C for 15 min. b Effect of temperature on ArLS activity. Reactions were conducted in 50 mM EPPS (pH 8.0) containing 0.1 mg mL⁻¹ enzyme, 50 μM GPP, and 5 mM Mg²⁺ across a temperature range of 10–50 °C for 15 min. c Effect of divalent metal ions on ArLS activity. Reactions were conducted in 50 mM EPPS (pH 8.0) containing 0.1 mg mL⁻¹ enzyme, 50 μM GPP and 1 mM of each metal ion at 30 °C for 15 min. d Effect of Mn²⁺ and Mg²⁺ concentrations on ArLS activity. Reactions were conducted in 50 mM EPPS (pH 8.0) containing 0.1 mg mL⁻¹ enzyme, 50 μM GPP, and various concentrations of Mn²⁺ or Mg²⁺ at 30 °C for 15 min. All experiments were conducted at least twice. Data represent the means of two independent experiments, with error bars indicating the range

To compare the catalytic efficiencies, we determined the kinetic parameters of ArLS and MsLS using GPP as the substrate and limonene production as the readout (Fig. S2). Substrate inhibition was observed at GPP concentrations above 50 µM, consistent with previous LS studies [41]. ArLS activity increased with rising GPP concentrations, and kinetic parameters were calculated for concentrations < 100 µM using Michaelis–Menten plots. ArLS exhibited a more than 13-fold lower K_M value for GPP (K_M–ArLS = 5.5 ± 0.8 µM, and K_M–MsLS = 72 ± 9.0 µM), indicating significantly higher substrate affinity (Table 1). Although the k_cat of MsLS was approximately 2.4-fold higher (k_cat–ArLS = 2.2 ± 0.07 min⁻¹; k_cat–MsLS = 5.5 ± 0.3 min⁻¹), ArLS demonstrated a 5.4-fold greater catalytic efficiency (k_cat/K_M; Table 1). This study presents the first detailed kinetic analysis of ArLS, highlighting its superior catalytic properties compared to previously reported LSs. The markedly low K_M value of ArLS suggests a substantial advantage for microbial limonene production, facilitating efficient catalysis at low substrate concentrations.

Table 1.

Biochemical properties of characterized limonene synthases

	ArLS	MsLS	LaLS	MpLS	RnLS	CsLS	ClLS
Organism	Agastache rugosa	Mentha spicata	Lavandula angustifolia	Mentha piperita	Ricciocarpos natans	Cannabis sativa	Citrus limon
Optimal pH	8.0 a	-	7.0 b	6.7 c	6.5 d	6.5 b	7.0 d
Optimal temperature	30 °C a	-	30 °C b	-	-	40 °C b	-
Optimal cofactor	Mn2+ (0.1 mM a)	-	Mg2+ (50 mM b)	Mn2+ (1 mM c)	Mg2+ (10 mM d)		Mn2+ (0.6 mM d)
KM (µM)	5.5 ± 0.3 a	72 ± 9.0 a	47 ± 3.8 b	1.8 c	1.25 d	6.8 ± 2.6 b	0.7 d
kcat (min−1)	2.2 ± 0.07 a	5.5 ± 0.3 a	0.72 b	-	-	-	-
kcat/KM (min−1 ·µM−1)	0.4 ± 0.06 a	0.08 ± 0.01 a	0.015 b	-	-	-	-
Ref.	This study	This study	[36]	[49]	[52]	[38]	[41]

^a Determined via GC–FID, ^b Determined via GC–MS, ^c Analyzed via GLC and radio-GLC, ^d Analyzed via LSC

Homology modeling and site-directed mutagenesis of limonene synthase from Agastache rugosa active site

To elucidate the structural determinants of substrate–metal ion interactions, we conducted de novo structural modeling of ArLS using AlphaFold2 (Fig. 4) [50]. The predicted structure model indicated that ArLS comprises 31 α-helices and forms a dimeric assembly, consistent with the native molecular mass determined by GPC (Fig. 2c). Superimposing the ArLS model onto the crystal structure of MsLS (PDB ID: 2ONG) revealed a highly conserved α-helical fold (Fig. 4a). Cavity analysis demonstrated that the active site is positioned near the Mn²⁺-binding center, similar to MsLS (Fig. 4b) [51]. The previously reported MsLS crystal structure was determined in complex with 2-fluorolinalyl diphosphate (FLPP), a catalytic intermediate derived from GPP during cyclization. Hence, in this study, FLPP was utilized to facilitate a direct structural comparison with the reference structure and elucidate the binding mode of the substrate-derived intermediate within the active site. Within the active site pocket, 19 residues were predicted to contribute to substrate positioning and metal coordination, with 15 residues being highly conserved between ArLS and MsLS. Notably, four residues (Ser335, Ile359, Val467, and Val472) exhibited differences in the deeper hydrophobic region.

Fig. 4.

AlphaFold-based structural prediction of ArLS. a Structural alignment of the predicted in silico ArLS model (orange) with (S)-limonene synthase from MsLS (PDB ID: 2ONG, green). The catalytic site is shown as a black dashed circle. b Residues forming the substrate-binding pocket of ArLS (orange) superimposed on MsLS (green) in complex with FLPP (navy stick) and manganese ions (purple spheres). Atom coloring: phosphorus, orange; oxygen, red; and fluorine, silver. The binding pocket residues of each enzyme are shown as lines. c Catalytic residues of ArLS. FLPP is shown at the center (navy–orange sticks). The hydrophilic region contains the metal cluster (purple spheres) and the metal-binding motifs (teal), which coordinate the substrate and initiate ionization. The hydrophobic region consists of nonpolar residues (gray), including key aromatic side chains. Predicted hydrogen bonds are indicated using black dashed lines

To further investigate substrate binding, an ArLS–GPP–Mn²⁺ complex model was generated by superimposing MsLS onto the ArLS structure. The residues of the DDXXD and NSE/DTE motifs (Asp366, Asp370, Asp510, Thr514, and Asp523) are located in close proximity to Mn²⁺ and the diphosphate group (Fig. 4c). This observation indicates their potential involvement in Mn²⁺ coordination and electrostatic or hydrogen bond interactions with the diphosphate moiety in the hydrophilic region. In the hydrophobic region, residues W338, H593, I359, V467, V472, and S335 are positioned to interact with the hydrocarbon tail of GPP through hydrophobic interactions, aiding in substrate capture and stabilization of the cationic intermediate during catalysis. The functional roles of these residues were validated with alanine-scanning mutagenesis of all 19 predicted catalytic residues (Fig. S3–S4). Substitution of residues directly involved in metal coordination or GPP binding resulted in loss of activity, while substitution of peripheral residues caused a partial decrease in limonene production. Only S335A (91.5% of wild-type activity) and V467A (73.5%) maintained significant catalytic activity, whereas all other variants exhibited activities below 15% (Table 2). These findings underscore the critical roles of these conserved residues in substrate binding, metal coordination, and the stabilization of carbocation intermediates in ArLS, consistent with their proposed functions in other limonene synthases.

Table 2.

Specific activities of ArLS and its variants

ArLS variants	Specific activity (nmol min−1 mg−1)
WT	13.43 ± 0.044
D366A	N.A. a
D370A	N.A. a
D510A	N.A. a
T514A	N. A. a
D523A	0.29 ± 0.019
R329A	N.A. a
W338A	N.A. a
I359A	0.73 ± 0.015
I362A	0.77 ± 0.007
Y587A	N.A. a
H593A	N.A. a
S335A	12.29 ± 0.41
T363A	0.32 ± 0.014
Y441A	0.18 ± 0.005
S466A	0.55 ± 0.045
V467A	9.88 ± 1.43
V472A	1.93 ± 0.12
R507A	0.16 ± 0.002
K526A	0.53 ± 0.030

^a N.A., No detectable activity

Bioconversion of limonene from geranyl pyrophosphate

In E. coli strains engineered with the heterologous MVA pathway, the pool of IPP and DMAPP, precursors of GPP, reached approximately 30 µM g⁻¹ DCW following IPTG induction, while intracellular GPP remained below 0.5 µM g⁻¹ DCW [53]. This minimal intracellular GPP concentration represents a significant bottleneck for monoterpene biosynthesis [54] due to the strong influence of GPPS expression levels on monoterpenoid production. Therefore, a high substrate affinity is essential for efficient enzymatic performance in microbial systems. The catalytic efficiency under physiologically relevant conditions was assessed by measuring limonene production at low GPP concentrations. Each LS (5 mg mL⁻¹) was incubated with 1–10 µM GPP in the presence of the optimal metal cofactor at 30 °C for 15 min. Both MsLS and ArLS exhibited concentration-dependent increases in limonene formation, with ArLS consistently yielding higher levels than MsLS (Fig. 5). At 1 µM GPP, ArLS produced 0.66 µM limonene, achieving a 66.6% conversion rate, which was 1.15-fold higher than that of MsLS. These results suggest that ArLS significantly enhances product yield, highlighting its potential for application in microbial cell factories, particularly for efficient substrate turnover at low intracellular metabolite concentrations.

Fig. 5.

Limonene production at various substrate concentrations from LSs. Experiments were conducted to evaluate the limonene production capacity of ArLS compared with MsLS. ArLS reactions were conducted in 50 mM EPPS buffer (pH 8.0) containing 5 mg mL⁻¹ enzyme and 0.1 mM Mn²⁺ at 30 °C for 15 min. The MsLS reactions were conducted in 50 mM PIPES buffer (pH 6.7) containing 5 mg mL⁻¹ enzyme and 1 mM Mn²⁺ at 30 °C for 15 min. Data represent the means of two independent experiments, with error bars indicating the range. *p < 0.05 by two-tailed Welch’s t-test

Discussion

The high limonene content of Agastache rugosa indicates the presence of a potent LS [44]. While earlier research has documented the expression of this enzyme in Yarrowia lipolytica [55], its catalytic and structural characteristics are yet to be characterized. This study provides the first comprehensive characterization of ArLS, unveiling its elevated catalytic efficiency, strong substrate affinity and distinctive structural attributes, and underscoring its promise for biocatalysis and metabolic engineering applications.

The ArLS gene was cloned and expressed in E. coli, showing 71.45% sequence identity with the well-characterized MsLS. Amino acid sequence alignment also revealed two conserved metal-binding motifs commonly found in monoterpene cyclases: the “aspartate-rich” DDXXD motif (Asp366 to Asp370) and the NSE/DTE motif (Asp510 to Glu518; Fig. 2a). ArLS has a molecular mass of 66.1 kDa and forms dimers (Fig. 2b and c). This finding is notable given that previous studies, including structural analysis of MsLS, have indicated that monoterpene cyclases primarily function as monomers in solution. Although earlier studies have noted a dimeric arrangement in the crystal structure of MsLS, this could be attributed to crystallization artifacts or transient dimerization [51].

To further investigate the potential functional relevance of dimerization, size-exclusion chromatography was conducted under various pH conditions (Fig. S5). The native dimeric species (132 kDa) were most prevalent at pH 8.0, while their abundance decreased at pH 9.0 and 10.0. At these higher pH values, where activity was diminished or eliminated, an additional species with an apparent molecular mass of 105 kDa emerged, indicating partial dimer dissociation under alkaline conditions. Our results offer experimental evidence for a stable dimeric state in solution that aligns with optimal enzymatic activity, suggesting a potential link between dimerization and the functional stability of ArLS. However, the direct causal relationship between the oligomeric state and catalytic function remains to be established. The functional implications of dimerization has been extensively documented in related terpene synthases, such as bornyl diphosphate synthase from Salvia officinalis, which forms a crucial homodimer for catalytic activity [56]. Future investigations utilizing X-ray crystallography or small-angle X-ray scattering (SAXS) are necessary to unveil the structural importance of ArLS dimerization.

ArLS displayed typical biochemical characteristics of plant-derived LS, including an optimal pH of 8.0, a moderate temperature preference (30 °C), and Mn²⁺ dependence, indicating alignment with physiological conditions (Table 1). The assay conditions for MsLS were selected based on those reported for limonene synthase from Mentha × piperita, which shares a high sequence identity (93%) with MsLS [49]. Among the limonene synthases with complete kinetic data, ArLS also showed high substrate affinity (K_M = 5.5 µM), significantly lower than MsLS (72 µM) and LaLS (47 µM), reflecting strong substrate-binding capability (Table 1). Although ArLS had a lower k_cat (2.2 min⁻¹) than MsLS (5.5 min⁻¹), it exhibited the highest catalytic efficiency (k_cat/K_M) among previously characterized LSs. This favorable k_cat/K_M suggests that ArLS can efficiently convert substrate even under limited intracellular GPP availability, a common bottleneck in microbial metabolic engineering. Overall, the standard reaction conditions and enhanced kinetic efficiency suggest that ArLS possesses a well-optimized catalytic structure, making it a promising candidate for high-efficiency monoterpene biosynthesis in engineered microbial systems.

Although ArLS demonstrated superior substrate affinity compared to MsLS, the difference in limonene production between the two enzymes was modest across the tested GPP concentrations (1–10 µM). This relatively small margin can be partially attributed to the higher k_cat of MsLS (5.5 min⁻¹) compared to ArLS (2.2 min⁻¹), which likely compensated for its lower substrate affinity under the given reaction conditions, thereby offsetting ArLS’s kinetic advantage in overall product yield. Nonetheless, ArLS consistently outperformed MsLS in terms of limonene yield across all tested concentrations, indicating a reproducible kinetic advantage under low-substrate conditions and supporting its potential as an effective biocatalyst, particularly when intracellular GPP availability is limited.

Structural alignment using AlphaFold revealed that ArLS maintains a highly conserved α-helical core and active site cavity compared to MsLS, supporting an evolutionarily conserved catalytic mechanism. Despite this similarity, the hydrophobic pocket’s core differed by only four residues (Ser335, Ile359, Val467, and Val472), corresponding to Cys321, Asn345, Ile453, and Met458 in MsLS. A previous study showed that substituting these four residues in MsLS with their ArLS equivalents (C321S/N345I/I453V/M458V) shifted product stereoselectivity from 96.8% (S)-limonene to 80.2% (R)-limonene [57], highlighting the importance of S335, I359, V467, and V472 in determining the enantioselectivity of ArLS. Conserved residues at positions 335 and 359 in (S)-limonene synthase were Cys or Thr and Asn or Cys, respectively, while Ser at 335 and Ile at 359 were characteristic of (R)-limonene synthase (Table S2) [58]. ArLS was classified as an (R)-limonene synthase based on its residue pattern and stereospecific product profile, consistent with its exclusive production of (R)-limonene [45]. Further investigation, including X-ray crystallography or cryo-EM of the ArLS–GPP–Mn²⁺ complex and molecular dynamics simulations, is needed for a comprehensive understanding of substrate affinity.

The functional characterization of the active-site residues presented here provides a basis for rational mutagenesis strategies to improve the stereoselectivity and catalytic productivity of ArLS. Future studies should utilize these findings to engineer ArLS variants with enhanced (R)-limonene specificity or increased k_cat, with potential applications in microbial cell factories for terpenoid biosynthesis. Ongoing endeavors in enzyme engineering and regulatory mechanisms may facilitate sustainable large-scale terpene production and advanced applications in industrial biotechnology.

Conclusion

In this study, we successfully characterized an ArLS and demonstrated its higher substrate affinity and limonene bioconversion efficiency compared to previously studied LSs. ArLS was robustly expressed in E. coli and maintains high catalytic activity under mild reaction conditions, underscoring its potential suitability for sustainable, scalable, and cell-based industrial production. Structural analysis of ArLS revealed key catalytic residues and subtle active-site differences compared to MsLS, which may underlie the conformational factors in enantioselective product formation. Our findings demonstrate that ArLS displays exceptional substrate affinity and catalytic efficiency among known LSs, positioning it as a promising enzyme candidate for further development for (R)-limonene production. This study broadens the enzymatic repertoire of limonene biosynthesis and provides valuable insights for future efforts to improve the economic feasibility of microbial limonene production.

Abbreviations

GPP

Geranyl pyrophosphate

IPP

Isopentenyl diphosphate

DMAPP

Dimethylallyl diphosphate

MEP

Methylerythritol-4-phosphate

MVA

Mevalonate

MsLS

Limonene synthase from Mentha spicata

LaLS

Limonene synthase from Lavandula angustifolia

StLS

Limonene synthase from Schizonepeta tenuifolia

CsLS

Limonene synthase from Cannabis sativa

PfLS

Limonene synthase from Perilla frutescens

CuLS

Limonene synthase from Citrus unshiu

ClLS

Limonene synthase from Citrus limon

RnLS

Limonene synthase from Ricciocarpos natans

AgLS

Limonene synthase from Abies grandis

PaLS

Limonene synthase from Picea abies

ArLS

Limonene synthase from Agastache rugosa

PCR

Polymerase chain reaction

LB

Luria–Bertani

SDM

Site-directed mutagenesis

EPPS

N-(2-hydroxyethyl)piperazine-N’-(3-propanesulfonic acid)

PIPES

Piperazine-N, N’-bis(2-ethanesulfonic acid)

CHES

2-(cyclohexylamino)ethanesulfonic acid

GPC

Gel permeation chromatography

GC–FID

Gas chromatography–flame ionization detection

GC–MS

Gas chromatography–mass spectrometry

GLC

Gas liquid chromatography

LSC

Liquid scintillation counting

LS

Limonene synthase

IPTG

Isopropyl-β-d-thiogalactopyranoside

SDS–PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

Authors’ contributions

SYJ and YJJ conceived, designed, and collected data. SYJ, YJJ, MJS, JY, BHS, and SJY analyzed the data and drafted the manuscript. All authors reviewed and approved the final version of the manuscript for publication.

Funding

This work was supported by the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (MSIT), through grants for the Enzyme Engineering for Next-Generation Biorefinery (NRF-2022M3J5A1056169 and NRF-2022M3J5A1085239), the Bio & Medical Technology Development Program (RS-2024-00440681 and RS-2025-24523548), the Research Initiative Program of KRIBB (NRF-2021M3A9I5023254), and the Yangyoung Foundation.

Data availability

All data generated or analyzed in this study are fully included within this article and its supplementary materials.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.