Sesquiterpene Synthase Research Articles

Crystal Structure of Caryolan-1-ol Synthase, a Sesquiterpene Synthase Catalyzing an Initial Anti-Markovnikov Cyclization Reaction.

In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from Streptomyces griseus, with and without an inactive analog of the farnesyl diphosphate (FPP) substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP structure was solved to 2.65 Å resolution and showed the ligand in an elongated orientation, incapable of undergoing the initial cyclization event to form a C1-C11 bond. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case, well fit by a curled-up tetraethylene glycol molecule recruited, presumably, from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotation of some active-site residues in comparison to the 2FFPP-structure. Most notably, W56 and F183 undergo 90° rotations between the 2FFPP complex and apoenzyme structures, suggesting that these residues provide interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L and F183A variants were observed to be severely restricted in their ability to catalyze C1-C11 cyclization of the FPP substrate and instead produced predominantly acyclic terpene products dominated by farnesol, β-farnesene, and nerolidol.

Expanding the “Terpenome”: Applications of Unspecific Peroxygenases (UPOs) in Oxidations of Unnatural Terpenoids

AbstractAn unnatural tricyclic oxaterpenoid, obtained by treatment of the sesquiterpene synthase presilphiperfolan‐8β‐ol synthase (BcBOT2) with an unnatural farnesyl pyrophosphate ether derivative, itself obtained by chemical synthesis, was converted in oxidation studies as part of a broad screening program by selected unspecific peroxygenases (UPOs). Product analysis revealed that Agrocybe aegerita UPO, its mutant PaDa‐I, and two commercial ones, UPO54 and UPO49, provided new oxidation products with sufficient efficiency for subsequent upscaling that allowed product isolation and structure elucidation. As such new terpene‐based oxiranes and hemiacetals were formed by UPO‐mediated epoxidations and CH‐activation. The structure elucidation was further supported by comparison with products generated by chemical oxidation.

Functional characterization of seven terpene synthases from Ophiopogon japonicus via engineered Escherichia coli

Abstract Background Ophiopogon japonicus (L.f) Ker-Gawl. growing in Zhejiang is recognized as the Dao-di medicinal herb for the production of Ophiopogonis Radix. Borneol-7-O-[β-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside, a prominent pharmacologically active compound, serves as a marker distinguishing O. japonicus in Zhejiang from those in other geographical areas. It is synthesized from borneol through glycosylation, with terpene synthase (TPS) being the critical enzyme catalyzing the conversion of terpene precursors into borneol. Objective The aim of the study was to define key genes involved in biosynthesis of borneol in O. japonicus. Methods The candidate terpene synthase genes were identified from the root and leaf transcriptome data of O. japonicus in Zhejiang and the functions of these enzymes were identified using engineered Escherichia coli. Results This study developed a rapid expression system for monoterpene and sesquiterpene synthases based on engineered E. coli. Seven terpene synthase genes (OjTPS1 to OjTPS7) were identified in different terpene synthase subfamilies, including 2 from TPS-a, 4 from TPS-b, and 1 from TPS-g. Biochemical analysis using an engineered system E. coli demonstrated that all the 7 terpene synthases produced monoterpenes, and OjTPS3, OjTPS5, and OjTPS6 also yielded sesquiterpenes. Conclusions These 7 terpene synthases produced 13 monoterpenes and eight sesquiterpenes. Notably, OjTPS1 produced borneol establishing the groundwork for elucidating the biosynthetic pathways of borneol-7-O-[β-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside and other volatile oil components.

Z/E configuration controlled by a Taxus sesquiterpene synthase facilitating the biosynthesis of (3Z,6E)-α-farnesene

Plant enzymes often present advantages in the synthesis of natural products with specific configurations. Farnesene is a pharmacologically active sesquiterpene with three natural Z/E configurations, among which the enzyme selectively responsible for the biosynthesis of (3Z,6E)-α-farnesene remains elusive. Herein, a sesquiterpene synthase TwSTPS1 biosynthesizing (3Z,6E)-α-farnesene as the major product was identified from Taxus wallichiana through genome mining. Utilizing molecular dynamics simulations and mutation analysis, the catalytic mechanism of TwSTPS1, especially Z/E configuration control, was explored. Moreover, the crucial residues associated with the specific catalytic activity of TwSTPS1 was elucidated through mutagenesis experiments. The findings contribute to our understanding of the Z/E configuration control by plant terpene synthases and also provide an alternative tool for manipulating (3Z,6E)-α-farnesene production using synthetic biology.

Genome-wide identification of the histone modification gene family in Aquilaria sinensis and functional analysis of several HMs in response to MeJA and NaCl stress

Histone modifications (HMs) play various roles in growth, development, and resistance to abiotic stress. However, HMs have been systematically identified in a few plants, and identification of HMs in medicinal plants is very rare. Aquilaria sinensis is a typical stress-induced medicinal plant, in which HMs remain unexplored. We conducted a comprehensive study to identify HMs and obtained 123 HMs. To conduct evolutionary analysis, we constructed phylogenetic trees and analyzed gene structures. To conduct functional analysis, we performed promoter, GO, and KEGG analyses and ortholog analyses against AtHMs. Based on the expression profiles of different tissues and different layers of Agar-Wit, some HMs of A. sinensis (AsHMs) were predicted to be involved in the formation of agarwood, and their response to MeJA and NaCl stress was tested by qRT-PCR analysis. By analyzing the enrichment of H3K4me3, H3K27me3, and H4K5ac in the promoter regions of two key sesquiterpene synthase genes, AsTPS13/18, we hypothesized that AsHMs play important roles in the synthesis of agarwood sesquiterpenes. We confirmed this hypothesis by conducting RNAi transgenic interference experiments. This study provided valuable information and important biological theories for studying epigenetic regulation in the formation of agarwood. It also provided a framework for conducting further studies on the biological functions of HMs.

The Recent Progress of Tricyclic Aromadendrene-Type Sesquiterpenoids: Biological Activities and Biosynthesis.

The tricyclic-aromadendrene-type sesquiterpenes are widely distributed and exhibit a range of biological activities, including anti-inflammatory, analgesic, antioxidant, antibacterial, insecticidal and cytotoxic properties. Several key sesquiterpene synthases (STSs) of this type have been identified, of which, viridiflorol synthase has been engineered for efficiently biosynthesizing viridiflorol in an Escherichia coli strain. This paper comprehensively summarizes the distribution and biological activity of aromadendrene-type sesquiterpenes in plant essential oils and microorganisms. The progress in aromadendrene-type sesquiterpene biosynthesis research, including the modifications of key STSs and the optimization of synthetic pathways, is reviewed. Finally, the prospects and associated challenges for the application and biosynthesis of these natural products are also discussed.

The chromosome-level genome assembly of Cananga odorata provides insights into its evolution and terpenoid biosynthesis.

Cananga odorata is known as a natural perfume tree of the Annonaceae family in Magnoliales. However, its phylogenetic position and the molecular mechanisms involved in the biosynthesis of the floral volatile organic compounds (VOCs) remain unclear. Here, by combining a variety of sequencing platforms, we present a telomere-to-telomere (T2T) genome of C. odorata with 735.83 Mb, which represents the highest integrity and assembly quality of genome in magnoliid plants reported to date. Phylogenetic analysis based on multiple datasets and approaches showed that C. odorata, as a member of magnoliids, is sister to eudicots, after their divergence from monocots. Metabolomic of VOCs in the essential oil and flowers scent showed that sesquiterpenes, especially β-caryophyllene, were the major compounds. Two CoTPS21 homologues derived from tandem duplication events were highly expressed during flower development and were identified as the key sesquiterpene synthases for the production of β-caryophyllene. In addition, CoSPL3 and CoSPL9 were considered as potential transcription factors for activating the expression of CoTPS21 homologues. Our results shed light on the molecular mechanisms underlying the biosynthesis of the unique floral fragrance in C. odorata and provide new insights into the phylogenetic position of magnoliids.

Biosynthesis of bridged tricyclic sesquiterpenes in Inula lineariifolia.

Presilphiperfolane-type sesquiterpenes represent a unique group of atypical sesquiterpenoids characterized by their distinctive tricyclic structure. They have significant potential as lead compounds for pharmaceutical and agrochemical development. Herein, we utilized a transcriptomic approach to identify a terpene synthase (TPS) gene responsible for the biosynthesis of rare presilphiperfolane-type sesquiterpenes in Inula lineariifolia, designated as IlTPS1. Through phylogenetic analysis, we have identified the evolutionary conservation of key motifs, including RR(x)8W, DDxxD, and NSE/DTE in IlTPS1, which are shared with other tricyclic sesquiterpene synthases in the TPS-a subfamily of Asteraceae plants. Subsequent biochemical characterization of recombinant IlTPS1 revealed it to be a multiproduct enzyme responsible for the synthesis of various tricyclic sesquiterpene alcohols from farnesyl diphosphate (FPP), resulting in production of seven distinct sesquiterpenes. Mass spectrometry and nuclear magnetic resonance (NMR) spectrometry identified presilphiperfolan-8β-ol and presilphiperfol-7-ene as predominant products. Furthermore, biological activity assays revealed that the products from IlTPS1 exhibited a potent antifungal activity against Nigrospora oryzae. Our study represents a significant advancement as it not only functionally identifies the first step enzyme in presilphiperfolane biosynthesis but also establishes the heterologous bioproduction of these unique sesquiterpenes.

Comprehensive Engineering Strategies for Heterologous Production of Zealexin A1 in Saccharomyces cerevisiae.

Zealexin A1 is a nonvolatile sesquiterpene phytoalexin, which not only exhibits extensive antifungal and insecticidal activities but also has the ability to enhance the drought resistance of plants, and thus has potential applications in agricultural and food fields. In this study, the biosynthetic pathway of zealexin A1 was constructed in Saccharomyces cerevisiae for the first time, and the highest production of zealexin A1 reported to date was achieved. First, through screening of sesquiterpene synthases from various plants, BdMAS11 had a stronger (S)-β-macrocarpene synthesis ability was obtained, and the heterologous synthesis of zealexin A1 was achieved by coexpressing BdMAS11 with cytochrome P450 oxygenase ZmCYP71Z18. Subsequently, after the site-directed mutagenesis of BdMAS11, fusion expression of farnesyl diphosphate synthase ERG20 and BdMAS11, and tailored truncation of BdMAS11 and ZmCYP71Z18, the strain coexpressing the manipulated BdMAS11 and original ZmCYP71Z18 produced 119.31 mg/L of zealexin A1 in shake-flask fermentation. Finally, the production of zealexin A1 reached 1.17 g/L through fed-batch fermentation in a 5 L bioreactor, which was 261.7-fold that of the original strain. This study lays the foundation for the industrial production of zealexin A1 and other terpenoids.

Single Point Mutation Abolishes Water Capture in Germacradien-4-ol Synthase.

The high-fidelity sesquiterpene cyclase (-)-germacradien-4-ol synthase (GdolS) converts farnesyl diphosphate into the macrocyclic alcohol (-)-germacradien-4-ol. Site-directed mutagenesis was used to decipher the role of key residues in the water control mechanism. Replacement of Ala176, located in the G1/2 helix, with non-polar aliphatic residues of increasing size (valine, leucine, isoleucine and methionine) resulted in the accumulation of the non-hydroxylated products germacrene A and germacrene D. In contrast, hydroxylation was maintained when the polar residues threonine, glutamine or aspartate replaced Ala176. Additionally, although a contribution of His150 to the nucleophilic water addition could be ruled out, the imidazole ring of His150 appears to assist carbocation stabilisation. The results presented here shed light on how hydroxylating sesquiterpene synthases can be engineered to design modified sesquiterpene synthases to reduce the need for further steps in the biocatalytic production of oxygenated sesquiterpenoids.

Engineering a versatile yeast platform for sesquiterpene production from glucose or methanol.

Natural sesquiterpene are valuable compounds with diverse applications in industries, such as cosmetics and energy. Microbial synthesis offers a promising way for sesquiterpene production. Methanol, can be synthesized from CO2 and solar energy, serves as a sustainable carbon source. However, it is still a challenge to utilize methanol for the synthesis of value-added compounds. Pichia pastoris (syn. Komagataella phaffii), known for its efficient utilization of glucose and methanol, has been widely used in protein synthesis. With advancements in technology, P. pastoris is gradually engineered for chemicals production. Here, we successfully achieved the synthesis of α-bisabolene in P. pastoris with dual carbon sources by expressing the α-bisabolene synthase gene under constitutive promoters. We systematically analyzed the effects of different steps in the mevalonate (MVA) pathway when methanol or glucose was used as the carbon source. Our finding revealed that the sesquiterpene synthase module significantly increased the production when methanol was used. While the metabolic modules MK and PMK greatly improved carbon source utilization, cell growth, and titer when glucose was used. Additionally, we demonstrated the synthesis of β-farnesene from dual carbon source by replacing the α-bisabolene synthase with a β-farnesene synthase. This study establishes a platform strain that is capable to synthesize sesquiterpene from different carbon sources in P. pastoris. Moreover, it paves the way for the development of P. pastoris as a high-efficiency microbial cell factory for producing various chemicals, and lays foundation for large-scale synthesis of high value-added chemicals efficiently from methanol in P. pastoris.

Characterization of a novel cytosolic sesquiterpene synthase MpTPS4 from Mentha ×piperita as a bioresource for the enrichment of invaluable viridiflorol in mentha essential oil

Our research addresses the challenge of low concentrations of viridiflorol, a unique and highly valuable sesquiterpene found in various Mentha species. We employed biotechnological strategies to enhance viridiflorol production, which could significantly boost export revenue. Mentha piperita L. sesquiterpene synthase (MpTPS4) was the focus of our study because it is a key enzyme in the biosynthesis of viridiflorol. Through biochemical characterization, we confirmed that MpTPS4 exclusively synthesizes viridiflorol. By overexpressing MpTPS4 in M. ×piperita L. using a glandular trichome-specific promoter, we achieved a notable increase (9–25 %) in viridiflorol content. Additionally, we explored the practical application of viridiflorol as a deterrent against the herbivore Helicoverpa armigera. The RNAi-mediated knockdown of MpTPS4 resulted in a significant reduction in viridiflorol levels in the essential oil. More importantly, these results show how relevant MpTPS4 is for making viridiflorol and how biotechnology could be used to increase biosynthesis. Our research provides valuable insights into enhancing the production of this commercially important sesquiterpene, offering promising opportunities for the mentha industry.

Functional characterization of sesquiterpene synthase in Mongolian medicine Syringa oblata in heartwood formation

Lilac (Syringa oblata) is a well-known horticultural plant, and its aromatic heartwood is widely utilized in Traditional Mongolian Medicine for treating angina. However, limited research on the dynamic changes and mechanisms of aromatic substance formation during heartwood development hinders the analysis and utilization of its medicinal components. In this study, volatile metabolome analysis revealed that sesquiterpenes are the primary metabolites responsible for the aroma in heartwood, with cadinane and eremophilane types being the most prevalent. Among the identified sesquiterpene synthases, SoSTPS1-5 exhibited significantly increased expression in heartwood formation and was selected for further investigation. Molecular docking simulations predicted multiple amino acid binding sites and confirmed its ability to catalyze the formation of eremophilane, copaene, cadinane, germacrane, and elemane-type sesquiterpenes from FPP (farnesyl pyrophosphate). Co-expression and promoter analysis suggested a transcriptional regulatory network primarily involving WRKY transcription factors. Additionally, aiotic and biotic stress inducers, such as Ag+, Fusarium oxysporum, and especially MeJA, were found to activate the expression of SoSTPS1-5 and promote sesquiterpene accumulation. This study provides insights into the basis of medicinal substance formation and the potential mechanisms of sesquiterpene accumulation in lilac heartwood, laying a foundation for future research on the biosynthesis and utilization of its medicinal components.

The role of CitMYC3 in regulation of valencene synthesis-related CsTPS1 and CitAP2.10 in Newhall Sweet Orange

CsTPS1 (Cs5g12900) is a gene encoding a sesquiterpene synthase involved in generating valencene, a key volatile compound in sweet orange fruit. Here, a citrus basic helix-loop-helix (bHLH) transcription factor (CitMYC3), is characterized as a positive regulator of CsTPS1. Dual-luciferase assays showed that CitMYC3 transcriptionally activates its own promoter, also the CsTPS1 promoter and, in addition, the promoter of a second transcription factor, CitAP2.10, which in turn positively regulates the CsTPS1 expression. During the developmental period, CitMYC3 transcripts were detectable at 60 days after full bloom (DAFB) and increased gradually to 120 DAFB, while CsTPS1 mRNA rapidly increased from 90 to 150 DAFB. Exogenous ethylene accelerated the expression of CitMYC3 (peaking at 4 h) before CsTPS1 expression (at 24 h). Furthermore, CitMYC3 and CitAP2.10 were also shown to activate the promoter of Arabidopsis AtTPS11, a homolog of CsTPS1, which is in charge of the synthesis of over fifteen different sesquiterpenes, indicating that CitMYC3 and CitAP2.10 have a likely conserved function in the regulation of sesquiterpene genes across a variety of plant species.

Genome Mining of Fungal Unique Trichodiene Synthase-like Sesquiterpene Synthases.

Sesquiterpenoids served as an important source for natural product drug discovery. Although genome mining approaches have revealed numerous novel sesquiterpenoids and biosynthetic enzymes, the comprehensive landscape of fungal sesquiterpene synthases (STSs) remains elusive. In this study, 123 previously reported fungal STSs were subjected to phylogenetic analysis, resulting in the identification of a fungi-specific STS family known as trichodiene synthase-like sesquiterpene synthases (TDTSs). Subsequently, the application of hidden Markov models allowed the discovery of 517 TDTSs from our in-house fungi genome library of over 400 sequenced genomes, and these TDTSs were defined into 79 families based on a sequence similarity network. Based on the novelty of protein sequences and the completeness of their biosynthetic gene clusters, 23 TDTS genes were selected for heterologous expression in Aspergillus oryzae. In total, 10 TDTSs were active and collectively produced 12 mono- and sesquiterpenes, resulting in the identification of the first chamipinene synthase, as well as the first fungi-derived cedrene, sabinene, and camphene synthases. Additionally, with the guidance of functionally characterized TDTSs, we found that TDTSs in Family 1 could produce bridged-cyclic sesquiterpenes, while those in Family 2 could synthesize spiro- and bridged-cyclic sesquiterpenes. Our research presents a new avenue for the genome mining of fungal sesquiterpenoids.

Functional Characterization of Sesquiterpene Synthases and P450 Enzymes in Flammulina velutipes for Biosynthesis of Spiro [4.5] Decane Terpene.

Flammulina velutipes, a popular edible mushroom, contains sesquiterpenes with potential health benefits. We characterized 12 sesquiterpene synthases and one P450 enzyme in F. velutipes using Aspergillus oryzae as a heterologous expression system, culminating in the biosynthesis of 16 distinct sesquiterpene compounds. An enzyme encoded by the axeB gene responsible for the synthesis of the spiro [4.5] decane compound axenol was discovered, and the mechanism of spirocycle formation was elucidated through quantum mechanical calculations. Furthermore, we delineated the role of a P450 enzyme colocated with AxeB in producing the novel compound 3-oxo-axenol. Our findings highlight the diverse array of sesquiterpene skeletons and functional groups biosynthesized by these enzymes in F. velutipes and underscore the effectiveness of the A. oryzae system as a heterologous host for expressing genes in the Basidiomycota genome. These insights into the biosynthesis of bioactive compounds in F. velutipes have significant implications for functional food and drug development.

Catalytic Mechanism and Heterologous Biosynthesis Application of Sesquiterpene Synthases.

Sesquiterpenes comprise a diverse group of natural products with a wide range of applications in cosmetics, food, medicine, agriculture, and biofuels. Heterologous biosynthesis is increasingly employed for sesquiterpene production, aiming to overcome the limitations associated with chemical synthesis and natural extraction. Sesquiterpene synthases (STSs) play a crucial role in the heterologous biosynthesis of sesquiterpene. Under the catalysis of STSs, over 300 skeletons are produced through various cyclization processes (C1-C10 closure, C1-C11 closure, C1-C6 closure, and C1-C7 closure), which are responsible for the diversity of sesquiterpenes. According to the cyclization types, we gave an overview of advances in understanding the mechanism of STSs cyclization from the aspects of protein crystal structures and site-directed mutagenesis. We also summarized the applications of engineering STSs in the heterologous biosynthesis of sesquiterpene. Finally, the bottlenecks and potential research directions related to the STSs cyclization mechanism and application of modified STSs were presented.

Functional and Mechanistic Characterization of the 4,5-diepi-Isoishwarane Synthase from the Liverwort Radula lindenbergiana.

The microbial type sesquiterpene synthase RlMTPSL4 from the liverwort Radula lindenbergiana was investigated for its products, showing the formation of several sesquiterpene hydrocarbons. The main product was structurally characterized as the new compound 4,5-diepi-isoishwarane, while the side products included the known hydrocarbons germacrene A, α-selinene, eremophilene and 4,5-diepi-aristolochene. The cyclization mechanism towards 4,5-diepi-isoishwarane catalyzed by RlMTPSL4 was investigated through isotopic labeling experiments, revealing the stereochemical course for the deprotonation step to the neutral intermediate germacrene A, a reprotonation for its further cyclization, and a 1,2-hydride shift along the cascade. The absolute configuration of 4,5-diepi-isoishwarane was determined using a stereoselective deuteration approach, revealing an absolute configuration typically observed for a microbial type sesquiterpene.

RNA-seq analysis reveals narrow differential gene expression in MEP and MVA pathways responsible for phytochemical divergence in extreme genotypes of Thymus daenensis Celak

BackgroundHere, we investigated the underlying transcriptional-level evidence behind phytochemical differences between two metabolically extreme genotypes of Thymus daenensis. The genotypes ‘Zagheh-11’ (thymol/carvacrol type, poor in essential oil [EO] [2.9%] but rich in triterpenic acids) and ‘Malayer-21’ (thymol type and rich in EO [3.8%]) were selected from an ongoing breeding program and then clonally propagated for further experimental use.Materials and methodsGC-MS, GC-FID, and HPLC-PDA were utilized to monitor the fluctuation of secondary metabolites at four phenological stages (vegetative, bud burst, early, and full-flowering stages). The highest phytochemical divergence was observed at early flowering stage. Both genotypes were subjected to mRNA sequencing (approximately 100 million paired reads) at the aforementioned stage. The expression patterns of four key genes involved in the biosynthesis of terpenoids were also validated using qRT-PCR.ResultsCarvacrol content in ‘Zagheh-11’ (26.13%) was approximately 23 times higher than ‘Malayer-21’ (1.12%). Reciprocally, about 10% higher thymol was found in ‘Malayer-21’ (62.15%). Moreover, the concentrations of three major triterpenic acids in ‘Zagheh-11’ were approximately as twice as those found in ‘Malayer-21’. Transcriptome analysis revealed a total of 1840 unigenes that were differentially expressed, including terpene synthases, cytochrome P450, and terpenoid backbone genes. Several differentially expressed transcription factors (such as MYB, bZIP, HB-HD-ZIP, and WRKY families) were also identified. These results suggest that an active cytosolic mevalonate (MVA) pathway may be linked to higher levels of sesquiterpenes, triterpenic acids, and carvacrol in ‘Zagheh-11’. The chloroplastic pathway of methyl erythritol phosphate (MEP) may have also contributed to a higher accumulation of thymol in Malayer-21. Indeed, ‘Zagheh-11’ showed higher expression of certain genes (HMGR, CYP71D180, β-amyrin 28-monooxygenase, and sesquiterpene synthases) in the MVA pathway, while some genes in the MEP pathway (including DXR, ispG, and γ-terpinene synthase) were distinctly expressed in Malayer-21. Future efforts in metabolic engineering of MVA/MEP pathways may benefit from these findings to produce increased levels of desired secondary metabolites at commercial scale.

Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch.

Sesquiterpene synthases (STPSs) catalyze carbocation-driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation-reprotonation process is widely used in STPSs to promote structural diversity, largely attributable to the distinct regio/stereoselective reprotonations. However, the molecular basis for reprotonation regioselectivity remains largely understudied. Herein, we analyzed two highly paralogous STPSs, Artabotrys hexapetalus (-)-cyperene synthase (AhCS) and ishwarane synthase (AhIS), which catalyze reactions that are distinct from the regioselective protonation of germacrene A (GA), resulting in distinct skeletons of 5/5/6 tricyclic (-)-cyperene and 6/6/5/3 tetracyclic ishwarane, respectively. Isotopic labeling experiments demonstrated that these protonations occur at C3 and C6 of GA in AhCS and AhIS, respectively. The cryo-electron microscopy-derived AhCS complex structure provided the structural basis for identifying different key active site residues that may govern their functional disparity. The structure-guided mutagenesis of these residues resulted in successful functional interconversion between AhCS and AhIS, thus targeting the three active site residues [L311-S419-C458]/[M311-V419-A458] that may act as a C3/C6 reprotonation switch for GA. These findings facilitate the rational design or directed evolution of STPSs with structurally diverse skeletons.