Showing papers on "Terpene published in 2021"

Cannabinoids, Phenolics, Terpenes and Alkaloids of Cannabis.

Abstract: Cannabis sativa is one of the oldest medicinal plants in the world. It was introduced into western medicine during the early 19th century. It contains a complex mixture of secondary metabolites, including cannabinoids and non-cannabinoid-type constituents. More than 500 compounds have been reported from C. sativa, of which 125 cannabinoids have been isolated and/or identified as cannabinoids. Cannabinoids are C21 terpeno-phenolic compounds specific to Cannabis. The non-cannabinoid constituents include: non-cannabinoid phenols, flavonoids, terpenes, alkaloids and others. This review discusses the chemistry of the cannabinoids and major non-cannabinoid constituents (terpenes, non-cannabinoid phenolics, and alkaloids) with special emphasis on their chemical structures, methods of isolation, and identification.

Cannabis sativa terpenes are cannabimimetic and selectively enhance cannabinoid activity.

Abstract: Limited evidence has suggested that terpenes found in Cannabis sativa are analgesic, and could produce an “entourage effect” whereby they modulate cannabinoids to result in improved outcomes. However this hypothesis is controversial, with limited evidence. We thus investigated Cannabis sativa terpenes alone and with the cannabinoid agonist WIN55,212 using in vitro and in vivo approaches. We found that the terpenes α-humulene, geraniol, linalool, and β-pinene produced cannabinoid tetrad behaviors in mice, suggesting cannabimimetic activity. Some behaviors could be blocked by cannabinoid or adenosine receptor antagonists, suggesting a mixed mechanism of action. These behavioral effects were selectively additive with WIN55,212, suggesting terpenes can boost cannabinoid activity. In vitro experiments showed that all terpenes activated the CB1R, while some activated other targets. Our findings suggest that these Cannabis terpenes are multifunctional cannabimimetic ligands that provide conceptual support for the entourage effect hypothesis and could be used to enhance the therapeutic properties of cannabinoids.

In vitro neuroprotective potential of terpenes from industrial orange juice by-products

Abstract: Citrus sinensis (orange) by-products represent one of the most abundant citric residues from orange juice industrial production, and are a promising source of health-promoting compounds like terpenes. In this work, different extraction solvents have been employed to increase terpene extraction yield and selectivity from this orange juice by-product. A set of bioactivity assays including enzymatic (acetylcholinesterase (AChE), butylcholinesterase (BChE) and lipoxygenase (LOX)) as well as antioxidant (ABTS, reactive oxygen species (ROS) and reactive nitrogen species (RNS)) activity tests have been applied to investigate the neuroprotective potential of these compounds. New fluorescence-based methodologies were developed for AChE and BChE assays to overcome the drawbacks of these tests when used in vitro to determine the anticholinergic activity of colored extracts. Comprehensive phytochemical profiling based on gas chromatography coupled to quadrupole time of flight mass spectrometry (GC-qTOF-MS) analysis showed ahigh content of mono- and sesquiterpenes in the extracts obtained with ethyl acetate, whereas n-heptane extracts exhibited a large amount of triterpenes and carotenoids. From a neuroprotective activity point of view, ethyl acetate extract is the most promising due to its anticholinergic activity and antioxidant capacity. Finally, a multivariate data analysis revealed a good correlation between some monoterpenes (e.g. nerol or limonene) and the antioxidant capacity of the natural extract, while a group of sesquiterpenes (e.g.δ-Cadinene or nootkatone) showed correlation with the observed AChE, BChE and LOX inhibition capacity. Hydrocarbons mono- and sesquiterpenoids reveal high capacity in vitro to cross the blood-brain barrier (BBB).

Extraction of Phenolic Compounds and Terpenes from Cannabis sativa L. By-Products: From Conventional to Intensified Processes.

Abstract: Cannabis sativa L. is a controversial crop due to its high tetrahydrocannabinol content varieties; however, the hemp varieties get an increased interest. This paper describes (i) the main categories of phenolic compounds (flavonoids, stilbenoids and lignans) and terpenes (monoterpenes and sesquiterpenes) from C. sativa by-products and their biological activities and (ii) the main extraction techniques for their recovery. It includes not only common techniques such as conventional solvent extraction, and hydrodistillation, but also intensification and emerging techniques such as ultrasound-assisted extraction or supercritical CO2 extraction. The effect of the operating conditions on the yield and composition of these categories of phenolic compounds and terpenes was discussed. A thorough investigation of innovative extraction techniques is indeed crucial for the extraction of phenolic compounds and terpenes from cannabis toward a sustainable industrial valorization of the whole plant.

Grape terpenoids: flavor importance, genetic regulation, and future potential.

Abstract: Terpenes significantly affect the flavor and quality of grapes and wine. This review summarizes recent research on terpenoids with regard to grape wine. Although, the grapevine terpene synthase gene family is the largest identified, genetic modifications involving terpenes to improve wine flavor have received little attention. Key enzyme modulation alters metabolite production. Over the last decade, the heterologous manipulation of grape glycosidase has been used to alter terpenoids, and cytochrome P450s may affect terpene synthesis. Metabolic and genetic engineering can further modify terpenoid metabolism, while using transgenic grapevines (trait transfer to the plant) could yield more flavorful wine. We also discuss traits involved in wine aroma quality, and the strategies that can be used to improve grapevine breeding technology.

A Review of the Potential Use of Pinene and Linalool as Terpene-Based Medicines for Brain Health: Discovering Novel Therapeutics in the Flavours and Fragrances of Cannabis.

Abstract: "Medicinal cannabis" is defined as the use of cannabis-based products for the treatment of an illness. Investigations of cannabis compounds in psychiatric and neurological illnesses primarily focus on the major cannabinoids, cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC), which are hypothesised to benefit multiple illnesses manifesting cognitive impairment, neurodegeneration and neuro-inflammation, as well as chronic pain, epilepsy and post-traumatic stress disorder, respectively. The cannabis plant contains >500 compounds, including terpenes responsible for the flavour and fragrance profiles of plants. Recently, research has begun providing evidence on the potential use of certain plant-derived terpenes in modern medicine, demonstrating anti-oxidant, anti-inflammatory, and neuroprotective effects of these compounds. This review examined the effects of two key terpenes, pinene and linalool, on parameters relevant to neurological and psychiatric disorders, highlighting gaps in the literature and recommendations for future research into terpene therapeutics. Overall, evidence is mostly limited to preclinical studies and well-designed clinical trials are lacking. Nevertheless, existing data suggests that pinene and linalool are relevant candidates for further investigation as novel medicines for illnesses, including stroke, ischemia, inflammatory and neuropathic pain (including migraine), cognitive impairment (relevant to Alzheimer's disease and ageing), insomnia, anxiety, and depression. Linalool and pinene influence multiple neurotransmitter, inflammatory and neurotrophic signals as well as behaviour, demonstrating psycho-activity (albeit non-intoxicating). Optimising the phytochemical profile of cannabis chemovars to yield therapeutic levels of beneficial terpenes and cannabinoids, such as linalool, pinene and CBD, could present a unique opportunity to discover novel medicines to treat psychiatric and neurological illnesses; however, further research is needed.

Volatile terpenes – mediators of plant-to-plant communication

Abstract: Plants interact with other organisms employing volatile organic compounds (VOCs). The largest group of plant-released VOCs are terpenes, comprised of isoprene, monoterpenes, and sesquiterpenes. Mono- and sesquiterpenes are well-known communication compounds in plant-insect interactions, whereas the smallest, most commonly emitted terpene, isoprene, is rather assigned a function in combating abiotic stresses. Recently, it has become evident that different volatile terpenes also act as plant-to-plant signaling cues. Upon being perceived, specific volatile terpenes can sensitize distinct signaling pathways in receiver plant cells, which in turn trigger plant innate immune responses. This vastly extends the range of action of volatile terpenes, which not only protect plants from various biotic and abiotic stresses, but also convey information about environmental constraints within and between plants. As a result, plant-insect and plant-pathogen interactions, which are believed to influence each other through phytohormone crosstalk, are likely equally sensitive to reciprocal regulation via volatile terpene cues. Here, we review the current knowledge of terpenes as volatile semiochemicals and discuss why and how volatile terpenes make good signaling cues. We discuss how volatile terpenes may be perceived by plants, what are possible downstream signaling events in receiver plants, and how responses to different terpene cues might interact to orchestrate the net plant response to multiple stresses. Finally, we discuss how the signal can be further transmitted to the community level leading to a mutually beneficial community-scale response or distinct signaling with near kin.

Linalool Alleviates Aβ42-Induced Neurodegeneration via Suppressing ROS Production and Inflammation in Fly and Rat Models of Alzheimer's Disease.

Abstract: Terpenes are vital metabolites found in various plants and animals and known to be beneficial in the treatment of various diseases. Previously, our group identified terpenes that increased the survival of Alzheimer's disease (AD) model flies expressing human amyloid β (Aβ) and identified linalool as a neuroprotective terpene against Aβ toxicity. Linalool is a monoterpene that is commonly present as a constituent in essential oils from aromatic plants and is known to have anti-inflammatory, anticancer, antihyperlipidemia, antibacterial, and neuroprotective properties. Although several studies have shown the beneficial effect of linalool in AD animal models, the mechanisms underlying the beneficial effect of linalool on AD are yet to be elucidated. In the present study, we showed that linalool intake increased the survival of the AD model flies during development in a dose-dependent manner, while the survival of wild-type flies was not affected even at high linalool concentrations. Linalool also decreases Aβ-induced apoptosis in eye discs as well as the larval brain. Moreover, linalool intake was found to reduce neurodegeneration in the brain of adult AD model flies. However, linalool did not affect the total amount of Aβ42 protein or Aβ42 aggregation. Rather, linalool decreased Aβ-induced ROS levels, oxidative stress, and inflammatory response in the brains of AD model flies. Furthermore, linalool attenuated the induction of oxidative stress and gliosis by Aβ1-42 treatment in the rat hippocampus. Taken together, our data suggest that linalool exerts its beneficial effects on AD by reducing Aβ42-induced oxidative stress and inflammatory reactions.

Transcriptomics Integrated with Free and Bound Terpenoid Aroma Profiling during "Shine Muscat" (Vitis labrusca × V. vinifera) Grape Berry Development Reveals Coordinate Regulation of MEP Pathway and Terpene Synthase Gene Expression.

Abstract: Terpenes and their derivatives are important biomarkers of grape quality as they contribute to the flavor and aroma of grapes. However, the molecular basis of terpene biosynthesis throughout the grapevine phenological developmental cycle remains elusive. Our current study investigates the free and bound terpene biosynthesis of berries at different phenological stages from preveraison to harvest. Detailed gene expression (transcriptomics) analysis, terpenoid volatile production by gas chromatography-mass spectrometry (GC-MS), and in planta transient expression were employed. Our results show that concentrations of most individual terpenes at different stages are distinctive and increase from preveraison to the veraison stage followed by a decrease from veraison to maturity. The combined transcriptomic analysis and terpene profiling revealed that 22 genes belonging to the MEP pathway and multiple classes of transcription factor family members including bHLH and several hormone biosynthesis- or signaling-related genes likely participate in the regulation of terpenoid biosynthesis according to their specific expression patterns in berries. Quantitative real-time polymerase chain expression analysis of 8 key differentially expressed genes in MEP pathways and further 12 randomly selected genes was performed during 8 sampling stages and validated the RNA-seq-derived expression profiles. To further confirm the function of a subset of the differentially expressed genes, we investigated the effects of combined overexpression of 1-deoxy-d-xylulose-5-phosphate synthase (VvDXS1-LOC100249323), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (VvDXR-LOC100248516), and terpene synthase (VvTPS56-LOC100266449) on the production of terpenes by transient overexpression in Nicotiana benthamiana leaves. The overall developmental patterns of total terpenes and gene expression profiles will help guide the functional analyses of further candidate genes important for terpene biosynthesis of grape as well as identifying the master transcriptional and hormonal regulators of this pathway in the future.

Structure, catalysis, and inhibition mechanism of prenyltransferase

Abstract: Isoprenoids, also known as terpenes or terpenoids, represent a large family of natural products composed of five-carbon isopentenyl diphosphate or its isomer dimethylallyl diphosphate as the building blocks. Isoprenoids are structurally and functionally diverse and include dolichols, steroid hormones, carotenoids, retinoids, aromatic metabolites, the isoprenoid side-chain of ubiquinone, and isoprenoid attached signaling proteins. Productions of isoprenoids are catalyzed by a group of enzymes known as prenyltransferases, such as farnesyltransferases, geranylgeranyltransferases, terpenoid cyclase, squalene synthase, aromatic prenyltransferase, and cis- and trans-prenyltransferases. Because these enzymes are key in cellular processes and metabolic pathways, they are expected to be potential targets in new drug discovery. In this review, six distinct subsets of characterized prenyltransferases are structurally and mechanistically classified, including (1) head-to-tail prenyl synthase, (2) head-to-head prenyl synthase, (3) head-to-middle prenyl synthase, (4) terpenoid cyclase, (5) aromatic prenyltransferase, and (6) protein prenylation. Inhibitors of those enzymes for potential therapies against several diseases are discussed. Lastly, recent results on the structures of integral membrane enzyme, undecaprenyl pyrophosphate phosphatase, are also discussed.

Terpene toxicity and oxidative stress

Abstract: Terpene, extracted from a series of plants, animals, and fungi, is a widely studied class of chemicals which is divided into eight main subclasses, such as monoterpenes, diterpenes, triterpenes, and sesquiterpenes, based on carbon numbers and isoprene units. Most of the terpenes, particularly monoterpenes, have high cytotoxic potential shown in various model organisms, while some terpenes, such as β-caryophyllene, show antiinflammatory, antioxidant, and cytoprotective effects. α-terpineol and terpinolene are placed among the most toxic terpenes along with humulene and linalool. IC50/LC50 or LD50 value for each terpene is varying and time- and dose-dependent, in which the cytotoxic effect is primarily caused by plasma membrane disruption, lipid peroxidation, ROS production, MTP loss, and mitochondrial impairment. Liver, kidney, lungs, and neurological tissues are affected much more than other organs. In conclusion, each terpene, which is widely used as a food additive and/or in medicinal applications, should be addressed before any application to check whether it has potential acute or chronic harmful effects.

In Vitro Evaluation of the Activity of Terpenes and Cannabidiol against Human Coronavirus E229.

Abstract: The activity of a new, terpene-based formulation, code-named NT-VRL-1, against Human Coronavirus (HCoV) strain 229E was evaluated in human lung fibroblasts (MRC-5 cells), with and without the addition of cannabidiol (CBD). The main constituents in the terpene formulation used for the experiment were beta caryophyllene, eucalyptol, and citral. The tested formulation exhibited an antiviral effect when it was pre-incubated with the host cells prior to virus infection. The combination of NT-VRL-1 with CBD potentiated the antiviral effect better than the positive controls pyrazofurin and glycyrrhizin. There was a strong correlation between the quantitative results from a cell-viability assay and the cytopathic effect seen under the microscope after 72 h. To the best of our knowledge, this is the first report of activity of a combination of terpenes and CBD against a coronavirus.

Epoxidation of Terpenes

Abstract: Terpene epoxides are considered as potential primary intermediates in the synthesis of numerous green polymers including epoxy resins, polycarbonates, nonisocyanate polyurethanes and even some polyamides. In this chapter we describe recent efforts from our group to develop catalytic and noncatalytic processes for terpene epoxidation using a variety of oxidizing agents and process intensification methods. Most experimental tests deal with limonene epoxidation with applicability to some other terpenes also demonstrated.

Role of terpenes in plant defense to biotic stress

Abstract: Terpenes constitute a highly diverse class of chemical compounds produced by the plant and playing a role in many functions, both physiological and ecological. Plants constantly interact with other organisms, from beneficial to detrimental, and evolved sophisticated regulatory mechanisms to control these interactions. The role of terpenes, or more in general of terpenoids, as preformed or induced chemical deterrents to herbivores is known since long, and their complex role in plant defense against pathogens is increasingly present in the scientific literature. The synthesis of terpenes is one of the responses to attack in numerous plant-pathogen binomials, where terpenes act as specialized or generalized pathogen inhibitors. In this chapter, the involvement of terpenes and terpenoids in resistance to plant pathogens, such as fungi, bacteria, and viruses, and, where relevant, their vectors, is described.

The scarecrow-like transcription factor SlSCL3 regulates volatile terpene biosynthesis and glandular trichome size in tomato (Solanum lycopersicum)

Abstract: Tomato (Solanum lycopersicum L.) type VI glandular trichomes that occur on the surface of leaves, stems, young fruits and flowers produce and store a blend of volatile monoterpenes and sesquiterpenes. These compounds play important roles in the interaction with pathogens and herbivorous insects. Although the function of terpene synthases in the biosynthesis of volatile terpenes in tomato has been comprehensively investigated, the deciphering of their transcriptional regulation is only just emerging. We selected transcription factors that are over-expressed in trichomes based on existing transcriptome data and silenced them individually by virus-induced gene silencing. Of these, SlSCL3, a scarecrow-like (SCL) subfamily transcription factor, led to a significant decrease in volatile terpene content and expression of the corresponding terpene synthase genes when its transcription level was downregulated. Overexpression of SlSCL3 dramatically increased both the volatile terpene content and glandular trichome size, whereas its homozygous mutants showed reduced terpene biosynthesis. However, its heterozygous mutants also showed a significantly elevated volatile terpene content and enlarged glandular trichomes, similar to the overexpression plants. SlSCL3 modulates the expression of terpene biosynthetic pathway genes by transcriptional activation, but neither direct protein-DNA binding nor interaction with known regulators was observed. Moreover, transcript levels of the endogenous copy of SlSCL3 were decreased in the overexpression plants but increased in the heterozygous and homozygous mutants, suggesting feedback repression of its own promoter. Taken together, our results provide new insights into the role of SlSCL3 in the complex regulation of volatile terpene biosynthesis and glandular trichome development in tomato.

Fermentative production of enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis.

Abstract: Linalool, an acyclic monoterpene alcohol, is extensively used in the flavor and fragrance industries and exists as two enantiomers, (S)- and (R)-linalool, which have different odors and biological properties. Linalool extraction from natural plant tissues suffers from low product yield. Although linalool can also be chemically synthesized, its enantioselective production is difficult. Microbial production of terpenes has recently emerged as a novel, environmental-friendly alternative. Stereoselective production can also be achieved using this approach via enzymatic reactions. We previously succeeded in producing enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis, a member of the Enterobacteriaceae family of bacteria, via the heterologous mevalonate pathway with the highest linalool titer ever reported from engineered microbes. Here, we genetically modified a previously developed P. ananatis strain expressing the (S)-linalool synthase (AaLINS) from Actinidia arguta to further improve (S)-linalool production. AaLINS was mostly expressed as an insoluble form in P. ananatis; its soluble expression level was increased by N-terminal fusion of a halophilic β-lactamase from Chromohalobacter sp. 560 with hexahistidine. Furthermore, in combination with elevation of the precursor supply via the mevalonate pathway, the (S)-linalool titer was increased approximately 1.4-fold (4.7 ± 0.3 g/L) in comparison with the original strain (3.4 ± 0.2 g/L) in test-tube cultivation with an aqueous-organic biphasic fermentation system using isopropyl myristate as the organic solvent for in situ extraction of cytotoxic and semi-volatile (S)-linalool. The most productive strain, IP04S/pBLAAaLINS-ispA*, produced 10.9 g/L of (S)-linalool in “dual-phase” fed-batch fermentation, which was divided into a growth-phase and a subsequent production-phase. Thus far, this is the highest reported titer in the production of not only linalool but also all monoterpenes using microbes. This study demonstrates the potential of our metabolically engineered P. ananatis strain as a platform for economically feasible (S)-linalool production and provides insights into the stereoselective production of terpenes with high efficiency. This system is an environmentally friendly and economically valuable (S)-linalool production alternative. Mass production of enantiopure (S)-linalool can also lead to accurate assessment of its biological properties by providing an enantiopure substrate for study.

Potential Antioxidant Activity of Terpenes

Abstract: Terpenes play a key part in the metabolic processes of a wide variety of animals, plants and microorganisms in which they are produced. In nature, terpenoids serve a variety of purposes including defense, signaling and as key agents in metabolic processes. Terpenes have been used in perfumery, cosmetics and medicine for thousands of years and are still extracted from natural sources for these uses. Terpenes antioxidant activities may sometimes explain their capacity to adjust inflammation, immunological effects and neural signal transmission. They offer pertinent protection under oxidative stress situations including renal, liver, cancer, cardiovascular diseases, neurodegenerative and diabetes as well as in ageing mechanisms.

Extraction and Mass Spectrometric Characterization of Terpenes Recovered from Olive Leaves Using a New Adsorbent-Assisted Supercritical CO2 Process.

Abstract: This work reports the use of GC-QTOF-MS to obtain a deep characterization of terpenoid compounds recovered from olive leaves, which is one of the largest by-products generated by the olive oil industry. This work includes an innovative supercritical CO2 fractionation process based on the online coupling of supercritical fluid extraction (SFE) and dynamic adsorption/desorption for the selective enrichment of terpenoids in the different olive leaves extracts. The selectivity of different commercial adsorbents such as silica gel, zeolite, and aluminum oxide was evaluated toward the different terpene families present in olive leaves. Operating at 30 MPa and 60 °C, an adsorbent-assisted fractionation was carried out every 20 min for a total time of 120 min. For the first time, GC-QTOF-MS allowed the identification of 40 terpenoids in olive leaves. The GC-QTOF-MS results indicate that silica gel is a suitable adsorbent to partially retain polyunsaturated C10 and C15 terpenes. In addition, aluminum oxide increases C20 recoveries, whereas crystalline zeolites favor C30 terpenes recoveries. The different healthy properties that have been described for terpenoids makes the current SFE-GC-QTOF-MS process especially interesting and suitable for their revalorization.

Phenol group of terpenoids is crucial for antibacterial activity upon ion leakage

Abstract: Plant tissues are natural sources of bioactive molecules to control pathogens that cause infections in plants and humans, as they contain secondary metabolites with antibacterial activities. Especially, terpenoids are responsible for the protection of various plants against herbivores and pathogens. In this study, we studied the antibacterial activity of 12 different terpenoid or related structures in a comparative way and revealed that the phenolic terpenoids are superior to other substituted derivatives against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus bacteria. We also aimed to analyse effect of these compounds on the bacteria cell wall structure and maintenance of homeostasis. We observed an immediate loss of cell membrane integrity and ion leakage upon treatment of both Gram-negative and Gram-positive cells with phenolic terpenoids carvacrol and thymol. After prolonged treatment, leakage of genetic material and increase of membrane permeability for molecules were also observed. Hydroxyl group of the phenol moiety was essential for this effect, since neither the O-methyl derivatives nor the benzylic partners were as effective. We thus propose that perturbation of ion homeostasis upon increase in cell wall permeability is key for the action of terpenoids against bacteria cells.

Effects of four terpenes on the mortality of Ditylenchus dipsaci (Kühn) Filipjev

Abstract: Laboratory experiments were conducted to investigate the possible nematicidal effect of the four terpenes, carvacrol, geraniol, eugenol and thymol, against the stem and bulb nematode Ditylenchus dipsaci (Kuhn, 1857) Filipjev, Proceedings of the Helminthological Society of Washington, 3, 80–82, 1936, isolated from infested garlic cloves. In in vitro tests the observed nematicidal activity of terpenes in descending order was carvacrol > eugenol > geraniol > thymol. Carvacrol exhibited the highest activity of 100% mortality when it was tested at the concentration of 2000 μl/L. No synergistic action was recorded when all four terpenes were used in mixture. In soil experiments thymol showed the highest nematicidal activity amongst all the other terpenes followed by carvacrol. Our results indicate that there is a positive correlation between the concentration and the exposure time. In addition, increasing the dose of each terpene increased paralysis rates of the nematode were recorded. These results strongly show the nematicidal activity of terpenes, but further studies are needed to ascertain their mode of action against nematodes.

Terpene Cyclases and Prenyltransferases: Structures and Mechanisms of Action

Abstract: Terpenes and isoprenoids are the most diverse small organic molecules on Earth. Here, we obtained the structures of sesquisabinene and santalene synthases from Santalum album L. in the presence or ...

Lithokinetic therapy after SWL: the possibilities of natural terpenes in combination with vitamin E

Abstract: М.Ю. Просянников1, О.В. Золотухин2, Ю.Ю. Мадыкин3, О.В. Константинова1, Д.А Войтко1, Н.В. Анохин1, А.В. Сивков1 1 НИИ урологии и интервенционной радиологии им. Н.А. Лопаткина – филиал ФГБУ «НМИЦ радиологии» Минздрава России; д. 51, ул. 3-я Парковая, Москва, 105425, Россия 2 БУЗ ВО «Воронежская областная клиническая больница No1»; д. 151, Московский проспект, Воронеж, 394066, Россия 3 ФГБОУ ВО «Воронежский государственный медицинский университет имени Н.Н. Бурденко»; д. 4, ул. Вайцеховского, Воронеж, 394036, Россия Контакт: Просянников Михаил Юрьевич, prosyannikov@gmail.com

Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus.

Abstract: Linalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.