Introduction

Terpenoids are structurally diverse and are the most abundant natural products among the myriad of compounds produced by plants, with biological roles ranging from growth and development to intracellular signaling and defense against predatory species1. Applications of these valuable compounds in the industries include as pharmaceuticals, flavors, fragrances and biofuels2. In higher plants, terpenoids are synthesized via either the cytosolic mevalonate (MVA) pathway or the plastidial methylerythritol phosphate (MEP) pathway, where the precursors are converted into structurally diverse terpenoids by the family of terpene synthases (TPSs). Sesquiterpene synthases responsible for sesquiterpenes (C15) production are localized in the cytosol, whereas monoterpene synthases that catalyze the production of monoterpenes (C10) are present in the plastids. Monoterpene synthases (600–650 amino acids) are longer than sesquiterpene synthases (550–580 amino acids) due to their N-terminal signal peptides that target the initial translation products towards the plastid3. A number of plant monoterpene and sesquiterpene synthases of molecular masses ranging from 50 to 100 kDa (monomers or homodimers) have been isolated and characterized with similar properties such as requirement for a divalent metal ion, having pI value near 5.0 and pH optimum within a unit of neutrality4.

Despite the lack of significant sequence similarities, terpene synthases share highly conserved tertiary and quaternary structural features dominated by α-helical folds known as class I terpene synthase fold5,6. These proteins consist entirely of α-helices and short connecting loops and turns that are organized into two structural domains of a non-functional N-terminus and a catalytically active C-terminus7. The class I terpene synthases which include monoterpene and sesquiterpene synthases utilize a trinuclear magnesium cluster coordinated by two conserved metal-binding motifs (DDxxD and NSE/DTE) to initiate catalysis8. The trinuclear magnesium cluster facilitates orientation of the substrate diphosphate moiety in the active site and triggers substrate ionization that generates reactive carbocation intermediates which undergo a series of cyclization, hydride shifts or other arrangements until the reactions are terminated by protons loss or by the addition of water7,9. The ligand binding causes conformational changes that cap and sequester the active site, thereby protecting the reactive carbocation intermediates from premature quenching by bulk solvents5,8.

One of the most fascinating features of the terpene synthases group is its ability to form a single product or multiple products from a sole substrate4,7,10,11. Furthermore, some terpene synthases exhibit multi-substrate abilities by synthesizing terpenes of different chain lengths depending on the corresponding substrate availability12,13,14,15. The structural basis of fidelity and promiscuity of the terpene synthases is related to the contour of the active site that serves as a template for catalysis by ensuring substrates and intermediates bind in the proper conformations, thereby controlling the formation of final catalysis product(s)16,17,18. Accordingly, the active site contours are product-like especially for high fidelity synthases to ensure the generation of specific product(s)8.

Linalool/nerolidol synthase is a multi-substrate enzyme with the capability to use GPP or FPP as a substrate, leading to the synthesis of linalool or nerolidol, respectively. Linalool participates in a complex interplay between pollinator attraction and plant defense against herbivory by attracting natural enemies of the herbivores19,20. Similarly, nerolidol has been identified as a potent signal that induces accumulation of defense-related compounds with extensive natural anti-herbivore or anti-pathogen effects21,22. These compounds are widely used as fragrance materials in cosmetic products including perfumes, lotions and shampoos, and in non-cosmetic products such as detergents and cleansers. Isolation and characterization of this enzyme were reported from Plectranthus amboinicus15, Rosa chinensis14, Hedychium coronarium23, Vitis vinifera24 and Antirrhinum majus13 which showed that this type of bifunctional enzyme is widespread across multiple plant species. The multi-substrate activity may confer advantages on plants to adapt rapidly in response to changes in the substrate profile under perturbation of metabolism in stressed plants, as well as under certain developmental changes without compromising their central metabolism12.

In our previous study, a putative monoterpene synthase gene (PamTps1) was isolated from P. amboinicus and introduced into the E. coli Rosetta™ 2 (DE3), which resulted in the production of linalool and nerolidol. Functional characterization demonstrated that this multi-substrate enzyme predominantly catalyzed formation of linalool and nerolidol from GPP and FPP, respectively, and was designated as a linalool/nerolidol synthase (Accession no: QGN03393)15. To learn more about PamTps1, biochemical characterization such as pH dependence, temperature dependence, divalent metal ion and substrate preferences, and kinetic properties were investigated. A reliable 3D homology model depicting the conformation of the P. amboinicus linalool/nerolidol synthase and the position of both GPP and the FPP substrates in the active site were also predicted in this analysis. Identification of the key residues involved in the active site architecture and catalysis reaction were also conducted. This model will serve as a basis for protein engineering to improve this bifunctional synthase with regard to product specificity or catalytic efficiency, and as a guide to future exploitations of this enzyme in terpenoids production.

Results and discussion

Effects of pH and temperature on PamTps1 activity

The PamTps1 activity was investigated using GPP as a substrate over a pH range of 5.5 to 9.0. At pH 6.5, the maximum catalytic activity was observed but was reduced to less than 10% of the maximum activity at pH 5.5 and pH 9 (Fig. 1A). This result was similar to the 3R-linalool synthase of Mentha citrate which exhibited an optimum pH close to pH 6.5 and a half maximum velocity at pH 7.525. Typically, the optimal pH for terpene synthases is within a unit of neutrality as reviewed by Bohlmann et al.4. Previously characterized plant linalool synthases showed an optimal pH range of 6.0–8.023,26,27,28,29. It was also noted that monoterpene synthases had a pH optima of 6–7 that correlated with the pH of the chloroplast in plants30,31,32, which corroborated the findings of PamTps1. Solvolytic decomposition of GPP to linalool in the presence of divalent cation was reported to occur under acidic condition33. As a result, the effect of pH below 5.5 could not be determined accurately due to an increase of substrate decomposition to linalool, which was also observed by Crowell et al.25.

Figure 1
figure 1

Biochemical characterization of PamTps1. (A) pH; (B) Temperature; (C) Mg2+ concentrations; (D) Mn2+ concentrations. Michaelis–Menten plot of PamTps1 at different concentrations of (E) Mn2+; (F) Mg2+; (G) GPP and (H) FPP. The saturation curve was constructed using Michaelis–Menten equation by hyperbolic regression. Values were reported as the mean of relative activity ± SD of triplicate analysis.

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The enzymatic activity of PamTps1 was conducted at temperatures ranging from 25 to 50 °C. Optimal catalytic activity was observed at 30 °C, with only half of the maximal activity noted at 25 °C and 37 °C (Fig. 1B). The observed result was similar to the temperature range (30–40 °C) reported for plant terpene synthases such as ocimene synthase of Lilium26, linalool synthase of coriander34 and Hedychium coronarium23, cineole synthase of lavender35 and β-sesquiphellandrane synthase of Persicaria minor36. The catalytic activity of PamTps1 dropped drastically beyond the optimum temperature, with only less than 10% of the full velocity retained at 50 °C. This could probably be linked to the destabilization of the three-dimensional structure of the enzyme at higher temperatures and ultimately contributed to denaturation and irreversible loss of activity37.

Effects of divalent metals on PamTps1 activity

Terpene synthases have an absolute requirement for a divalent metal ion such as Mg2+ or Mn2+ as a cofactor. The role of divalent metal ion in terpene synthases catalysis has been widely discussed and presumably involved in both substrate binding and catalysis8,17. Chelation of metal ion such as Mg2+, neutralizes two of the three negative charges of the diphosphate moiety of the substrate, thereby assisting the ionization of the allylic substrate into highly reactive carbocation intermediates38. Thus, divalent metal ions preferences of PamTps1 and their influence on the catalytic activity were evaluated at different concentrations of Mg2+ (0–250 mM) and Mn2+ (0–10 mM).

In the absence of a divalent metal ion, the PamTps1 activity was negligent. However, the activity was restored by the provision of either Mg2+ or Mn2+, which suggested an absolute requirement for a metal ion cofactor for catalytic activity (Fig. 1C,D). A maximal activity was obtained with Mn2+ at 0.5 mM, but was inhibited as Mn2+ concentration increased to 10 mM (Fig. 1D). Other characterized plant terpene synthases demonstrated maximum activity with manganese concentrations at less than 1.0 mM28,39,40. On the other hand, in the presence of Mg2+, the catalytic activity of PamTps1 increased steadily from 2 mM to a maximum activity at 20 mM, but was inhibited at 250 mM (Fig. 1C). This optimal concentration of Mg2+ finding was also observed in M. citrata linalool synthase25, Citrus sinensis limonene synthase40 and Japanese pepper terpene synthases39. In this study, PamTps1 showed a preference for Mg2+ for catalysis with 2.1 folds increase in activity compared to Mn2+. Likewise, other characterized plant terpene synthases that favored Mg2+ over Mn2+ included linalool/nerolidol synthase 1 and 213, Artemisia annua monoterpene synthases41, Lilium ‘Siberia’ terpene synthase26 and Santalum album terpene synthases42. In contrast, linalool synthase of lavender28 and C. sinensis limonene synthase40 showed preferences for Mn2+ as a cofactor with high terpene yields when 1–5 mM of Mn2+ were used.

Kinetic parameters of PamTps1

In this study, PamTps1 activity was inhibited when Mg2+ and Mn2+ concentrations beyond 50 mM and 5 mM, respectively, were used. Therefore, the Km value was estimated by a non-linear Michaelis–Menten curve using lower concentrations of Mg2+ and Mn2+ (Fig. 1E,F) which gave 1.74 ± 0.35 mM and 0.05 ± 0.001 mM, respectively, (Table 1). These values were comparable to those obtained with kiwi terpene synthases43, snapdragon linalool/nerolidol synthase13 and sweet basil geraniol synthase44. Nevertheless, in some reported metal ions studies, there are other terpene synthases that recorded Km values of less than 1 mM23,27,45 while higher Km values were also noted in some terpene synthases including A. annua linalool synthase46 and γ-terpinene synthases10,47. Although Km value for PamTps1 was substantially lower when using Mn2+, its Vmax value was only 43% of that with Mg2+. It is presumed that PamTps1 is more likely to operate with Mg2+ cofactor in planta due to the higher concentration of Mg2+ in plant cells as compared to the Mn2+ 48,49.

Table 1 Kinetic properties of PamTps1.
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Kinetic characterization of PamTps1 for GPP and FPP was performed below 100 µM since higher concentrations inhibited its catalytic activity (Fig. 1G,H). The apparent Km value of PamTps1 for GPP was 16.72 ± 1.32 µM, which was well within the range of Km values reported in other plant monoterpene synthases (Table 1)40,41, but lower compared to linalool synthases from H. coronarium (20.54 ± 4.52 µM)23, Cinnamomum osmophloeum (54.19 µM)50, L. angustifolia (55.8 ± 4.1 µM)28 and M. citrata (25 ± 6.00 µM)25. Nonetheless, linalool synthases from snapdragon13, A. arguta27 and A. chinensis51 exhibited Km values below 10 µM which suggested that these enzymes have a higher affinity for GPP. On the other hand, PamTps1 Km value of 40.47 ± 3.83 µM for FPP was 2.4 folds higher than that for GPP, which signified that PamTps1 had a higher binding affinity for GPP.

From the abovementioned results, it can be inferred that PamTps1 has a lower affinity for FPP and become saturated at a higher substrate concentration to reach its maximal velocity (Vmax = 14.85 ± 2.80 µmol mg−1). PamTps1 has a greater affinity for GPP than FPP as anticipated for a monoterpene synthase, where a lower concentration of GPP was required to achieve Vmax of 24.16 ± 3.75 µmol mg−1. Similar observation was noted in the snapdragon linalool/nerolidol synthases that exhibited higher substrate affinity towards GPP than to FPP13. The turnover rate (kcat) for both substrates in the current study were 0.16 s−1 and 0.10 s−1 for GPP and FPP, respectively, which was within the range of monoterpene (0.01–1.0 s−1) and sesquiterpene (0.03–0.5 s−1) synthases recorded52,53, and the low kcat values reflected that PamTps1 is a relatively slow enzyme. Terpene synthases are typically slow enzymes, which is a general feature of the enzymes involved in secondary metabolism and is approximately 30 folds slower than those involved in central metabolism54. The catalytic efficiency (kcat/Km) of GPP was 3.9 folds higher than FPP, further suggesting that PamTps1 recognized GPP more efficiently, which was in accordance with the abovementioned expectations. This may also be linked to the fact that PamTps1 was a plastid-targeted enzyme, where the GPP pool was located. Parallel observations were seen in lavender28 and Freesia55.

Secondary structure prediction

The secondary structure of PamTps1 was predicted using PSIPRED server56 followed by identification and annotation of the protein domain using MOTIF and SMART57. The PSIPRED tool predicted that the secondary structure of PamTps1 would consist entirely of α-helices (24 α-helices) connected by coils, with no strands or β-sheets observed except for the two extended strands located at the N-terminal signal peptide region (Fig. S1). Through domain analysis, it was revealed that these α-helices were organized into two structural domains of N-terminal (residues: 66–245) (Pfam: PF01397) and C-terminal metal binding domain (residues: 277–540) (Pfam: PF03936) with domain boundary located at residue M271 as determined by DomPRED. These predictions are in agreement with general features of most plant terpene synthases that adopt an α-helical architecture, which are organized into two domains of N-terminal region that has structural similarity to glycosylhydrolases58 and the C-terminal domain containing the catalytic site5.

Protein homology modelling of the PamTps1

The PamTps1 was modeled on the crystal structure of Salvia officinalis (+)-bornyl diphosphate synthase (BPPS) (1N24)5 using residues that correspond to the complete amino acid sequence in accordance to the RRx8W motif. The chosen BPPS template featured a closed active site conformation with Mg2+ and its product, and shared 67.04% sequence identity. The residue numbers described hereafter corresponded to the numbering of amino acids immediately following the RRx8W motif (Fig. S2). The predicted PamTps1 structure as shown in Fig. 2, revealed that the enzyme comprised of two structural domains of N- and C-terminal, connecting with short loops and turns. The N-terminal domain (residues 1–214) of PamTps1 consisted of 14 α-helices arranged in an α-barrel with minor structural differences to that of BPPS5. Although there was no established catalytic function for this N-terminal domain, it was reported that this domain was involved in capping the active site pocket upon substrate binding, and presumably shielded the reactive carbocation intermediates from water as observed in the crystal structure of BPPS, Taxus brevifolia taxadiene synthase (PDB ID: 3P5R) and Gossypium arboretum δ-cadinene synthase (PDB ID: 3G4F)5,9,59. The presence of this apparently non-functional N-terminal domain in terpene synthases may have been due to an evolutionary vestige from copalyl diphosphate synthase-kaurene synthase, which was the ancestor of all modern terpene synthases that possess both functional catalytic domains6,8,60.

Figure 2
figure 2

Protein homology modelling of PamTps1 using SWISS-MODEL server showed the structural domains and the active site of the enzyme. (A) model structure of PamTps1 made up of α-helices with N-terminal domain (green) and C-terminal domain (blue). (B) Ribbon view of PamTps1 model. The helical segment was designated according to Tarshis et al.67. All conserved motifs were labelled in the figure and Mg2+ was illustrated as green spheres.

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The N-terminal domain contained two conserved motifs that were present in typical plant terpene synthases, namely the RRx8W and LQLYEASFLL motifs. The tandem arginine motif was found in many plant monoterpene synthases and was thought to mark the approximate cleavage site of the plastid-targeting sequence6. A previous truncation study of this motif from a limonene synthase suggested that the RR motif was required for initial isomerization of GPP to linalyl diphosphate (LPP), owing to the inability of the truncated limonene synthase to accept GPP as a substrate, while still functioning with LPP as a substrate for the cyclization step6,61. These arginine residues may also contribute to the stabilization of the closed active site while still allowing flexibility that was necessary for the binding of two structurally different prenyl diphosphates (GPP and LPP) as observed in limonene synthase6. Since PamTps1 did not undergo a cyclisation reaction, it was likely that the RRx8W motif might only be involved in the capping of the PamTps1 active site and not in the catalysis reaction. The InterProScan analysis also predicted that the RRx8W region acted as an active site lid in the PamTps1. Besides that, the LQLYEASFLL motif that was assumed to be part of the active site62,63 occurred as LQLYEASFLE in PamTps1, and there were no observable differences in the overall structure of the enzyme for amino acid substitution from leucine to glutamic acid.

The larger C-terminal domain (residues 215–542) adopted an α-helical architecture known as class I terpene synthase fold which consisted of 16 α-helices, where the hydrophobic pocket of the active site cavity was formed by six α-helices (C, D, F, G, H and J) (Fig. 2). This domain was well conserved with an RMSD value of 0.190 Å as compared to the BPPS. The C-terminal domain contained two metal binding motifs of the aspartate-rich DDxxD and NSE/DTE (evolved from a second aspartate-rich region) to form a consensus sequence of (L,V)(V,L,A)(N,D)D(L,I,V)x(S,T)xxxE. The NSE/DTE motif appeared to be less well conserved amongst the plant terpene synthases as compared to the DDxxD motif. Both the DDxxD and NSE/DTE motifs were reported to bind to a trinuclear magnesium cluster involved in the fixation of the diphosphate substrate5,64,65. The PamTps1 also contained other motifs that were thought to be part of the terpene synthases active site, such as RxR and GTLxEL63,66 which occurred as RDR and GTLDEL in PamTps1 and were located 35 amino acids upstream and two amino acids downstream of the DDxxD, respectively.

Protein structural alignment or superimposition allows homology establishment between template and protein model based on the 3D protein conformation as a protein structure was more conserved than its sequence during evolution. Superimposition of the PamTps1 model with BPPS template using Chimera with α-carbon RMSD fitted to 0.203 Å showed that the two structures were exceptionally similar (Fig. 3).

Figure 3
figure 3

Superimposition of PamTps1 model (purple) with BPPS template (brown) using Chimera. The α-carbon RMSD value of 0.203 Å indicated the two structures were exceptionally similar. The aspartate-rich motif was red, the DTE motif was orange and the green spheres were magnesium ions.

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Validation of the PamTps1 model

The reliability of the model was first evaluated by the GMQE and Qualitative Model Energy Analysis (QMEAN) scores provided by the SWISS-MODEL tool. The GMQE score is expressed as a number between 0 and 1, where higher numbers indicate higher reliability of the model68. The QMEAN Z-score provides an estimate degree of structural features similarity observed in the model with scores around 0 indicate good agreement between model structure and template69. The PamTps1 model scores of 0.82 and − 1.32 for respective GMQE and QMEAN showed that the built model was reliable and satisfactory. Further validation by PROCHECK to assess the stereochemical quality of generated model showed that 92.8% of PamTps1 residues fall in most favored regions, 6.6% residues in additional allowed regions, 0.2% residues in generously allowed regions and only 0.4% residues in the disallowed regions suggesting the acceptability of the modeled structure (Fig. S2, Table S3). PROVE analysis revealed that the quality of the predicted 3D structure of PamTps1 model was good and reliable with the respective Z-score mean and Z-score RMS for the entire structure of 0.487 and 1.421, respectively. The ERRAT analyses statistic of non-bonded interactions between different atom types based on characteristic atomic interactions97. The overall ERRAT quality factor value is expressed as the percentage of the protein for which the calculated value is less than the 95% rejection limit. A good high-resolution structure typically yields values of 95% or higher, and the PamTps1 model yielded an overall quality factor of 95.88%, which was very satisfactory. Another program used for validation of protein structure was the Verify3D, which determines compatibility of an atomic model (3D) with its own amino acid sequence (1D) by assigning structural class based on its location and environment98. The Verify3D analysis of PamTps1 model revealed that 95.73% of the residues had an average 3D–1D score ≥ 0.2. As the cut-off score ≥ 0, this implies that the predicted model was valid. ProSA was used to check the 3D model of PamTps1 for potential errors where positive value of the z-score corresponded to problematic or erroneous region of a model. The Z-score of − 12 for PamTps1 model was within the acceptable range of X-ray studies and this value was close to the value of the template (− 10.92) suggesting that the predicted model was reliable and close enough to experimentally determine structure (Fig. S2, Table S3).

Molecular docking of PamTps1 with prenyl diphosphate substrates

To gain further insight into the active site of the enzyme investigated here, the model structure of PamTps1 was carried out with molecular docking using GPP (C10) and FPP (C15) substrates. Docking of the prenyl diphosphate substrates yielded multiple docking positions. The criteria for choosing the best docking position were based on the lowest docking score and the number of hydrogen bonds between the substrate and the amino acid residues. A docking position with the least docking score has the highest affinity towards the ligand, and hence is the best docked conformation. Hydrogen bonds contribute to the stability of proteins and specificity of protein–ligand interactions, which is also an important consideration for selection of the docking position70. The docking results were further analyzed using Chimera and LigPlot + to generate 2D and 3D ligand–protein interaction diagrams, respectively.

Docking of GPP and FPP substrates confirmed that the active site of PamTps1 was located at the C-terminal domain, proximate to the location of the Mg2+ cofactor (Fig. 4). A two-dimensional representation of Mg2+ interaction with the amino acid residues and substrate (ligand) was displayed in Fig. 4C,D. This concurred with earlier observations using SWISS-MODEL and InterProscan that the diphosphate (PPi) moiety of the prenyl substrates interacted with the highly conserved aspartate-rich (D296DVYD300) and NSE/DTE (LAD440DLGT444APFE448) motifs via complexed Mg2+, in which the boldface residues were coordinated to the metal ions. The first and third aspartate residues in the aspartate-rich motif, D296 and D300, were coordinated to Mg2+A and Mg2+C, which were identical to the BPPS, avian FPP synthase67, taxadiene synthase9, and M. spicata limonene synthase6. The second metal-binding region comprised of D440, T444 and E448 of the helix H coordinated to the Mg2+B. Similar metal ion coordination by the corresponding residues was also observed in trichodiene synthase71, 5-epi-aristolochene synthase72 and taxadiene synthase9. The distances between Mg2+ cofactor and the corresponding residues were summarized in Table S1. The ideal distance for metal ion coordination was between 2.0 and 2.2 Å, which was more typically observed in higher-resolution structures73. It was revealed that the coordination distance with the metal ion for PamTps1 was within the range of 2.0–2.75 Å, which was longer than what was expected for Mg2+ coordination. Shorter metal–ligand distances resulted in tighter first coordination sphere ligands, resulting in less wiggle room in the first coordination sphere, and therefore less deviation from the ideal octahedral geometry74. Magnesium has the tightest initial coordination sphere closest to ideal octahedral geometry, with a typical Mg—O distance of around 2.1 Å74. Validation of metal-binding sites of PamTps1 revealed that two of the three metal ions exhibited octahedral geometry, while the third had an outlier geometry (Table 2). The gRMSD measures overall deviation of the observed geometry angle from the ideal geometry angle75, and PamTps1 model showed acceptable gRMSD values for the trinuclear magnesium cluster binding sites. The vacancy calculates percentage of vacant coordination sites for a given geometry75. This analysis, however, revealed borderline and outlier vacancy values, which probably explained the longer metal coordination distances between magnesium ion and binding sites as discussed previously (Table S1).

Figure 4
figure 4

Ligand-PamTps1 interactions. Three-dimensional (3D) view of (A) GPP and (B) FPP docking at PamTps1 active site. The red–orange balls and stick chains represent PPi, green spheres are the Mg2+, the red side chains are the DDxxD motif, the orange side chains are the DTE motifs, the blue side chains are the hydrogen bond donor residues, the magenta side chains represent the aromatic residues and the cyan lines are the hydrogen bonds. Two-dimensional (2D) view of (C) GPP and (D) FPP docking at PamTps1 active site. Hydrogen bonds are shown as green dotted lines, Mg2+ are shown in green spheres and spoked arcs represent residues making non-bonded contacts with the hydrophobic tail of the ligand. Details of the docking result are summarized in Table S2.

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Table 2 Metal-binding site geometry analysis.
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In addition to metal coordination interactions, the PPi moiety of GPP and FPP were also predicted to accept hydrogen bonds from R259, R437 and K456 residues (Fig. 4; Table S2). Similarly, this finding was observed in other reported plant terpene synthases where PPi binding was accommodated by hydrogen bonds donated from two arginine and one lysine residues5,8,72. The R259 of PamTps1 derived from the R259DR motif may serve as a proton donor to thermodynamically support the PPi cleavage by protonation after the first reaction step76,77,78. Mutational analysis of this residue showed a loss of catalytic activity suggesting the important role of this arginine residue in restricting the PPi79. The R437 derived from the extended second metal binding motif (LR437LADDLGTAPFE) in PamTps1 was also reported to donate hydrogen bond to the PPi of the substrate as observed with the bornyl diphosphate synthase5. The K456 residue of the PamTps1 that was a part of the conserved lysine residue amongst Tpsb terpene synthases was located at the H-α1 loop and hydrogen bonded with the PPi of the substrate. The H-α1 loop lysine residue was also observed to donate hydrogen bond to the PPi in the BPPS crystal structure5 and limonene synthase6. The coordination of three metal ions and hydrogen bond interactions with basic residues of lysine and/or arginine presumably triggered the ionization of the substrate to yield carbocation intermediates that led to the production of terpenoids80,81. The substrate coordination and distance with PamTps1 residues and Mg2+ are summarized in Table S2. Based on the proposed mechanism of 5-epi-aristolochene synthase72 and Abies grandis α-bisabolene synthase82, the metal-dependent ionization of the substrate resulted in the generation of a negatively charged PPi that was stabilized by Mg2+ ions and three basic residues, and which created a positively charged region that drew the PPi away from the carbocations in the hydrophobic active pocket. Thus, the three Mg2+ ions and the three basic residues served as the PPi recognition motif in the active site, allowing proper orientation of the substrate while activating the PPi to initiate ionization and catalysis8.

The active site of terpene synthases was also characterized by the presence of several aromatic residues crucial for the stabilization of the carbocation intermediates5,8,9,79. The docking results revealed that the non-polar hydrocarbon groups of GPP and FPP were buried in the hydrophobic area of the active site surrounded by aliphatic and aromatic residues (Fig. 4C,D). The C10 tail of the GPP formed hydrophobic interactions with W268, Y272, V289, T293, I397, T517 and Y523 residues. Meanwhile the W268, Y272, V289, V292, T293, I397, A398, A399, A402, L436 and Y523 residues participated in non-bonded interactions with the C15 group of the FPP. Among the active site residues, the non-polar hydrocarbon group of GPP and FPP were located in the aromatic pair’s area surrounded by residues Y523 of the J-K loop and W268 of the helix C at the bottom of the PamTps1 active site (Fig. 4A,B). Sequences comparison against other terpene synthases suggested that the W268 was a conserved residue, whereas the position equivalent to Y523 could be occupied by aromatic residues of histidine, phenylalanine or tyrosine as mutation of these residues resulted in catalytically impaired catalyst6,8,79,83. According to Brandt et al.76, the nature and position of these aromatic amino acid residues at the active site of terpene synthases determined the docking orientation of the intermediate prenyl cation and therefore product specificity. In amorphadiene synthase, the aromatic phenylalanine residue (residue in the same position of Y523 of PamTps1) was similarly involved in positioning of the FPP substrate in the active site, which subsequently stabilized the carbocation intermediates84. A similar observation was also reported by Zhang et al.85 with Nicotiana tabacum 5-epi-aristolochene synthase (TEAS) that catalyzed the cyclisation of FPP into bicyclic 5-epi-aristolochene. Mutational analysis of the aromatic amino acids proved the essential role of these residues in the active site for stabilization of the carbocation intermediates79,85. Positioning of GPP and FPP in the PamTps1 active site surrounded by these aromatic residues suggested that this docking analysis was rational and compatible with other crystal structures of terpene synthases.

Insights into the PamTps1 active site pocket

Not all terpene synthases have the ability to use multi-substrate. Steric limitations and configuration of the active site center and the overall protein stability contributed by the tertiary protein structure might rule out the use of multi-substrate12. The ability of terpene synthases to catalyze multiple substrates has been reported to be contributed by both size and residues of the active site pockets. In general, the active site pocket is slightly larger than the corresponding substrate and product, and size of the cavity is increasingly deeper and wider for increasingly longer chain products9,86. The active site of Streptomyces clavuligens linalool/nerolidol synthase (bLinS) has been shown to be large enough to accommodate sesquiterpene, which explained the fact that this enzyme recognized FPP as a substrate64. It was predicted that the active site pocket of PamTps1 was also large enough and unconstrained to accommodate FPP, resulting in nerolidol formation.

Using CASTp server and InterProScan analysis, the topographic features of the PamTps1 active site pocket containing the docked substrate was illustrated in Fig. 5 and amino acids that lined the pocket cavity were also identified (Table 3). Both substrates were appropriately docked in the PamTps1 active site cavity, thus enlightened the multi-substrate use ability of PamTps1. The active site cavity of PamTps1 was a deep hydrophobic pocket with a contour defined by numerous aliphatic and aromatic side chains made of six helices of C, D, F, G, H and J (Table 3; Fig. 5) similarly as described for the BPPS structure5. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity. This result was also supported by structural studies of other plant terpene synthases5,9,25,72. It was reported that arginine, phenylalanine, tyrosine, valine, tryptophan and isoleucine were the commonly observed amino acid residues at the catalytic site of the terpene synthases87, which was also observed in the PamTps1 active site. The presence of aromatic residue pairs (Y523 and W268) at the bottom of the active site did not appear to restrict the size of the active site, and the hydrocarbon group of FPP appeared to fit perfectly into the catalytic pocket, which may shed light on the possibility of PamTps1 accepting FPP as a substrate (Fig. 5). By analogy with the previous characterized enzymes, it was believed that the active site of PamTps1 was reasonably large and deep enough to accommodate both the GPP and FPP, resulting in the formation of linalool and nerolidol, respectively.

Figure 5
figure 5

The PamTps1 active site pocket. The overview of PamTps1 active site pocket from (A) top and (B) side views. Docking positions of (C) GPP and (D) FPP in PamTps1 active site cavity. The PamTps1 active site is a deep hydrophobic pocket consisting of C, D, F, G, H and J helices. The red–orange ball and stick chains represent the PPi of substrate, green spheres are the Mg2+, the orange side chains are the DTE motifs, the blue side chains are the hydrogen bond donor residues and the magenta side chains represent the aromatic residues.

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Table 3 Amino acid residues involved in the establishment of PamTps1 active site pocket and catalysis activity predicted using CASTp and InterProScan.
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Residues from the RRx8W motif, RDR motif, H-α1 and J-K loops were observed to act as a catalytic lid that closed the active site entrance upon substrate binding (Table 3). Structural comparison with BPPS showed that the J-K loop of PamTps1 was longer than the equivalent loop in BPPS. Sugiura et al.88 reported that the Backhousia citriodora linalool synthase had a long J-K loop and bulky amino acids around the active site that could partially inhibit water access to the active site, leading to the production of (−) linalool and minor amounts of myrcene and (−) limonene. Alignment of amino acids indicated that most Lamiaceae linalool synthases differed from other terpene synthases by a three-amino acid deletion at the –-K loop region, thereby resulting in a more open structure, allowing easier access of water during substrate ionization25,89. However, no amino acid deletions at the J-K loop region were observed in the PamTps1 and it was assumed that the longer J-K loop could lead to the more open structure of the enzyme. The crystal structure of bLinS was also reported to be relatively open, allowing the carbocation intermediate to attack nearby water and led to linalool production64. Although the crystal structures PamTps1’s open and closed active site conformations are not yet available, it is thought that PamTps1 does not undergo significant conformational changes between open and closed states, as observed with other linalool synthases25,64. As a result, the active site was more susceptible to water access, resulting in the premature released of the carbocationic intermediates and the production of acyclic linalool and nerolidol25,64,89. Besides that, other conserved motifs considered to be part of the active site were LQLYEASFLL and GTLxEL63,66. However, there was no computational evidence that both motifs were involved in the formation of the PamTps1 active site or in the catalysis reaction, as exhibited by the active site pocket analysis and protein docking studies.

Conclusion

PamTps1 was classified as a linalool/nerolidol synthase with the ability to convert GPP and FPP into acyclic linalool and nerolidol, respectively. The catalytic activity of this recombinant synthase was optimal at pH 6.5 and 30 °C in the presence of 20 mM Mg2+ as a cofactor, which was within the range of most reported terpene synthases. PamTps1 catalysis was still stimulated by Mn2+ at the optimal concentration of 0.5 mM in place of Mg2+, however the catalytic activity was decreased by 2.1 folds. The kinetic properties of PamTps1 were analyzed using Michaelis–Menten equation, which revealed that it had a higher binding affinity and catalytic efficiency for GPP rather than FPP, as anticipated for a monoterpene synthase located in the plastid where the GPP pool was accessible. The PamTps1 model structure was successfully constructed from its amino acid sequences using BPPS as a template, and this model will serve as a first glimpse into the structural insights of PamTps1 catalytic site as a linalool/nerolidol synthase. The P. amboinicus linalool/nerolidol synthase exhibited features of a class I terpene synthase fold made up of α-helices architecture that contain the N-terminal domain and a catalytic C-terminal domain. Based on the prior knowledge of the reaction mechanisms of other monoterpene/sesquiterpene synthases, it is hypothesized that a PamTps1 reaction mechanism begins with the metal-dependent ionization of the PPi moiety of respective GPP or FPP to form a geranyl cation or farnesyl cation. Assisting the metal ions in PPi complexation are the basic residues of R259, R437 and K456 that direct the PPi away from the active site after ionization. The addition of water to the cationic intermediate, followed by deprotonation, resulted in the formation of acyclic terpenoids linalool and nerolidol. The ability of PamTps1 to use multiple substrates was believed to be due to the enzyme’s active site that was large enough to accommodate larger substrate such as FPP, allowing water capture that caused premature termination and subsequent nerolidol formation. This model will serve as a framework for exploring the roles of active site residues in rational design to exchange the enzyme function between monoterpene and sesquiterpene synthase.

Materials and methods

Plant material

The leaves of P. amboinicus were collected from purchased plants which were maintained at the Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia in Selangor, Malaysia (3° 00′ 26.4″ N 101° 42′ 19.3″ E). Plant authentication was performed by a botanist, Dr. Shamsul Khamis, from the School of Environmental Science and Natural Resources, Universiti Kebangsaan Malaysia, Selangor, Malaysia.

Functional characterization of recombinant PamTps1

The P. amboinicus linalool/nerolidol synthase (designated as PamTps1) (GenBank accession no: MK050501) was previously isolated and functionally characterized by Ashaari et al.15. Enzyme assay was conducted in a 100 µl reaction containing assay buffer (10 mM Tris–HCl, pH 7.5, 10% (v/v) glycerol, 1 mM DTT, 0.1 mM NaWO4, 0.05 mM NaF), 10 mM MgCl2 and 3–5 µg of purified protein. The enzymatic reaction was initiated by addition of 27 µM of GPP or FPP (Sigma Aldrich, USA) and incubated at 30 °C for 30 min. The terpene products released into the headspace of the assay mixture were collected by solid phase micro extraction (SPME) with a 100 µm polydimethylsiloxane (PDMS) coated fiber (Supelco, USA) at 60 °C for 30 min. The adsorbed products were separated through Agilent HP-5MS column (30 m × 250 µm inner diameter × 0.25 µm film thickness) and analyzed using Agilent 7890A gas chromatograph equipped with Agilent 5975C quadrupole mass spectrometer (Agilent Technologies, Santa Clara, USA). The SPME fiber containing the volatile compounds was inserted into GC injection port and thermally desorbed at 250 °C for 15 min using splitless mode with helium as carrier gas at a flow rate of 1 ml/min. The oven temperature was initially maintained at 50 °C and gradually increased to 280 °C at a rate of 10 °C/min for 3 min. The temperature of the ion source and transfer line was set at 220 °C and 280 °C, respectively, and electron impact mass spectra was recorded at 70 eV ionization energy. All assay products were identified by comparison of the mass spectra to the NIST14 library database and by comparing the retention times and mass spectra to the authentic standards of (−) linalool and nerolidol (Sigma Aldrich, USA). Standard calibration curves were constructed using the pure standards with concentrations ranging from 10 to 1000 µg/ml in the same conditions as the assay reactions.

Optimum temperature and pH of PamTps1 were determined by assaying at various temperatures ranging from 25 to 37 °C and seven pH levels, respectively. The buffer systems used in this study were 2-(N-Morpholino) ethanesulfonic acid (MES) buffer (pH 5.5–6.5) and Tris–HCl buffer (pH 7.0–9.0). Divalent cation preferences and optimum concentrations were determined by assaying at different MgCl2 (0.0, 2.0, 4.0, 10.0, 20.0, 50.0, 100, 250 mM) and MnCl2 (0.0, 0.1, 0.2, 1.0, 5.0, 10.0 mM) concentrations. The substrate dependence of PamTps1 was studied by adding GPP or FPP with different concentrations ranging from 0 to 200 µM to the reaction mixture. The kinetic parameters Km, Vmax, kcat and kcat/Vmax values were determined by fitting the data to the Michaelis–Menten equation analyzed using GraphPad Prism8. Extracted total crude proteins from Rosetta 2 (DE3) E. coli cells carrying empty pET-32b(+) vector were used as a negative control in place of PamTps1. One unit (U) of activity was defined as the amount of enzyme required to produce 1 µmole enzymatic product per min per ml under standard conditions. Specific activity was defined as enzyme activity (U) per mg of protein.

Secondary structure and 3D structure prediction

The motifs and domains were identified using MOTIFinder Search (https://www.genome.jp/tools/motif/), SMART (Simple Modular Architecture Research Tool) (http://smart.embl-heidelberg.de/) and InterProScan90. Secondary structure and domain boundary were predicted using PSIPRED Protein Structure Prediction (PSIPRED v3.3)56 (http://bioinf.cs.ucl.ac.uk/psipred/) and Protein Domain Prediction (DomPred)91 (http://bioinf.cs.ucl.ac.uk/psipred/?dompred), respectively. The three-dimensional protein structure of PamTps1 model was constructed from the amino acid sequence using automated comparative protein modelling server SWISS-MODEL92 (https://swissmodel.expasy.org/) and visualized using UCSF Chimera v 1.13rc93. The template for building the 3D structure of PamTps1 was obtained from the SWISS-MODEL Template Library and the most homologous sequence was considered as a potential template for the homology modeling92. The structural superimposition and calculation of the root-mean-square deviations (RMSD) between the model and template were conducted via Chimera using the carbon alpha (Cα) fitting method.

Validation of the PamTps1 model

The 3D model was evaluated by SWISS-MODEL’s Global Model Quality Estimation (GMQE) and Qualitative Model Energy Analysis (QMEAN) function. Structural evaluation and stereochemical analysis was conducted with Ramachandran plot using RAMPAGE server94 (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php). The model was further subjected to the Structural Analysis and Verification Server v. 5.0 (SAVES) (http://servicesn.mbi.ucla.edu/SAVES/) which included PROCHECK95, PROVE (PROtein Volume Evaluation)96, ERRAT97 and Verify3D analysis98,99 to evaluate the reliability of the predicted protein structure. Problematic region of the model was identified using Protein Structure Analysis (ProSA) server (https://prosa.services.came.sbg.ac.at), a tool commonly used to check 3D model protein structures for potential errors100.

Molecular docking

Protein–ligand docking simulation was conducted using the SwissDock server101 with the ligand selected from the ZINC database102. The docking assays were run using default parameters and the results were viewed via the Chimera software. Hydrogen bond network and distance between ligand and active site residues were also analyzed using Chimera. Distances of the amino acid residues which interacted with Mg2+ were also calculated. Identification of amino acids surrounding the active site was conducted by searching for atoms within < 5 Å of the docked ligand. Validation of metal-binding site was conducted using CheckMyMetal server74 (https://cmm.minorlab.org/) to assess the geometry of the metal-binding site and the vacancy of the metal.

Active site pocket analysis

Predictions of the active site pocket and of the amino acid(s) that contributed to the pocket were conducted by applying the CASTp 3.0 server (Computed Atlas of Surface Topography of Proteins)103.

Ethical statement of research involving plants

The P. amboinicus that was used in this study was purchased from Petani Kota Nursery located at Dengkil, Selangor, Malaysia (2° 53′ 38.7″ N 101° 45′ 9.0″ E), and it is from cultivated origin. All the methodology and data collection comply with relevant institutional, national and international guidelines and legislation.