Oxidosqualene Cyclase Research Articles

Identification, functional characterization and expression profiling of three triterpene synthases from the legume plant Vigna unguiculata

Oxidosqualene cyclases (OSCs) are important regulatory enzymes involved in cyclization reactions of 2, 3-oxidosqualene to form triterpenes and sterols. This study presents the identification and characterization of three OSC genes, a β – amyrin synthase (VuβAS), a lupeol synthase (VuLUS) and a cycloartenol synthase (VuCAS) in Vigna unguiculata, an edible leguminous plant with high nutritional and nutraceutical value. Phylogenetic analysis showed that the VuβAS, VuLUS and VuCAS were clustered within the clades of previously characterized β – amyrin synthases, lupeol synthases and cycloartenol synthases. Heterologous expression in Saccharomyces cerevisiae and Gas Chromatography – Mass Spectrometry (GC – MS) analysis in different plant stages confirmed their specific functions. VuβAS showed higher expression in roots from early germinating seedlings to older plants (4-day to 28-day), while VuLUS expression levels were higher in the roots of older plants only (14-day to 28-day). VuCAS expression was increased in all the tissues of 4-day seedlings, with a peak in stem and leaves and a lower accumulation in radicles. These findings revealed the presence and function of OSC genes in V. unguiculata, and future research could lead to the discovery of promising biologically active compounds.

Genome-wide identification and expression analysis of the OSC gene family in Platycodon grandiflorus

Platycodon grandiflorus stands as one of the most extensively utilized traditional Chinese medicinal herbs, with triterpenoids and their derivatives serving as its primary medicinal components. Oxido squalene cyclase (OSC), serving as a crucial enzyme in the triterpenoid synthesis pathway, has the capability to enzymatically generate significant quantities of sterols and triterpenoid intermediates. While the OSC gene family has been identified in numerous species, bioinformatics research on this family remains scant. Presently, the specific members of this gene family in Platycodon grandiflorus have yet to be definitively determined. In this study, we successfully identified a total of 15 PgOSC genes within the genome of Platycodon grandiflorus by conducting homology comparisons. These genes were discovered to be unevenly distributed across the five chromosomes of the species, organized in the form of gene clusters. Subsequently, we conducted a thorough analysis of the OSC gene family’s evolutionary relationship by constructing a phylogenetic tree. Other characteristics of PgOSC family members, including gene structure, conserved motifs, protein three-dimensional structure, subcellular localization, and cis-acting elements were thoroughly characterized. Furthermore, We analyzed the expression of PgOSC gene in different tissues of Platycodon grandiflorus by qRT-PCR, and found that the expression of PgOSC genes in root was higher than that in stem and leaf. Upon comparing the effects of salt, heat, and drought treatments, we observed a significant induction of PgOSC gene expression in Platycodon grandiflorus specifically under salt stress conditions. In summary, this study comprehensively identified and analyzed the OSC gene family, aiming to provide basic biological information for exploring the members of PgOSC gene family.

7076 Inhibiting Oxidosqualene Cyclase, An Enzyme In The Cholesterol Biosynthetic Pathway, Suppresses Lung Metastasis Of Triple-Negative Breast Cancer Cells

Abstract Disclosure: Y. Liang: None. S.M. Hyder: None. Triple-negative breast cancers (TNBC) lack ER, PR and Her2neu proteins that are commonly targeted in breast cancer therapies. As a consequence, women who suffer from TNBC have poor prognosis and limited treatment options, and are usually administered toxic, non-specific chemotherapeutic agents. Drug-resistance almost always occurs, leading to tumor metastasis, the main cause of patient death. New non-toxic therapeutic strategies to control metastasis of TNBC are urgently needed. Cholesterol is an essential structural and functional component of cell membranes that is necessary for the growth of both primary tumors and metastatic colonies that reside in distant organs. Therefore, inhibiting cholesterol production is an attractive therapeutic strategy. Treatment with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently, we targeted oxidosqualene cyclase (OSC), an enzyme that occurs downstream of HMGCoA-reductase in the cholesterol biosynthetic pathway, to disrupt tumor metastasis. Potent small molecule inhibitors of OSC have been identified. RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating several forms of primary tumors. We developed a mouse model for studying lung metastasis using a MDA-MB-231 variant (LM2) TNBC cell line obtained from Dr. Massagué (Memorial Sloan-Kettering Cancer Center). These are aggressive cells, and injection of only 2 X 105 cells produces detectable lung metastatic colonies in approximately 4-6 weeks. We inoculated female nu/nu mice, 5-6 weeks old, with 2 X 105 MDA-MB-231/LM2 cells in 0.1 ml DMEM/F12 medium without serum via tail vein. Five days after tumor cell inoculation animals received 20 mg/kg (n=6), 40 mg/kg (n=7) RO (ip) or vehicle alone (n=7) every other day for two weeks. Animals were then sacrificed, and both lungs harvested and immediately fixed in Bouin’s fixative. Metastatic colonies on the lung surface were identified and counted under a dissecting microscope. Our study showed that RO effectively reduced lung metastasis without affecting animal weight. Compared with 56 + 7 colonies (Mean ± SEM) in controls given vehicle alone, RO significantly reduced lung metastatic colonies to 28 ± 5 in animals exposed to 20 mg/kg and 6 ± 1 in 40 mg/kg treated animals (p<0.05, ANOVA). Reduction in number of colonies in 40 mg/kg group was also significantly different from colonies counted in 20 mg/kg treated animals. Our study is the first to demonstrate that disruption of cholesterol biosynthesis in a model of human TNBC leads to reduced metastasis in lungs. Further studies will determine mechanisms through which RO suppresses TNBC metastasis to the lungs. Supported by Zalk Missouri Professor Endowment Funds. Presentation: 6/3/2024

12347 Cholesterol Biosynthesis Inhibitor Ro 48-8071 Suppresses Growth Of Triple-negative Breast Cancer Cells By Inducing Apoptosis And Suppressing Angiogenesis

Abstract Disclosure: Y. Liang: None. S.M. Hyder: None. Triple-negative breast cancers (TNBC) lack ER, PR and Her2neu, proteins that are commonly targeted in breast cancer therapies. Women who suffer from TNBC have limited treatment options and are administered toxic chemotherapeutic agents. They have poor prognosis due to the emergence of drug-resistant cells that lead to tumor metastasis. New non-toxic therapeutic strategies to control TNBC progression, and prevent metastasis are urgently needed. Cholesterol is an essential structural and functional component of cell membranes necessary for tumor growth and progression. Therefore, inhibiting cholesterol production is an attractive therapeutic strategy since tumor cells grow rapidly, and need a greater supply of cholesterol. We targeted oxidosqualene cyclase (OSC), an enzyme that occurs downstream of HMGCoA-reductase in the cholesterol biosynthetic pathway, to disrupt tumor growth. RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating several forms of primary tumors but its effect on TNBC is not known. Pharmacological doses of RO potently reduced the viability of both MDA-MB-231 and BT20 TNBC cells in a dose dependent manner (1-50 μM). IC50 values for loss of cell viability was approximately 10 μM for both cell lines following 48 h exposure to RO. Doses close to the IC50 for OSC (1-10 nM) also reduced cell viability following exposure of TNBC cells to RO for a week. Importantly, RO had no effect on the viability of normal human mammary cells. FACS analysis revealed that RO dose-dependently induced apoptosis of TNBC cell lines in vitro. In vivo, administration of 5-10 mg/kg RO to mice with BT-20 TNBC cell-derived xenografts (100 mm3) inhibited tumor growth with no apparent toxicity. Immunohistochemical analysis of tumor tissues at the end of treatment showed that RO increased TUNEL expression, suggesting that the in vivo effects of RO were also exerted through mechanisms affecting apoptosis. Furthermore, IHC of tumor sections showed that RO also reduced levels of the angiogenic markers VEGF and CD-31 (blood vessels) in vivo, indicating that suppression of angiogenesis is also part of the mechanism through which RO exerts its anti-tumor effects. We conclude that RO is a potent inhibitor of TNBC and that its anti-tumor effects involve mechanisms that induce apoptosis and suppress angiogenesis. Presentation: 6/2/2024

7076 Inhibiting Oxidosqualene Cyclase, an Enzyme in the Cholesterol Biosynthetic Pathway, Suppresses Lung Metastasis of Triple-negative Breast Cancer Cells

Abstract Disclosure: Y. Liang: None. S.M. Hyder: None. Triple-negative breast cancers (TNBC) lack ER, PR and Her2neu proteins that are commonly targeted in breast cancer therapies. As a consequence, women who suffer from TNBC have poor prognosis and limited treatment options, and are usually administered toxic, non-specific chemotherapeutic agents. Drug-resistance almost always occurs, leading to tumor metastasis, the main cause of patient death. New non-toxic therapeutic strategies to control metastasis of TNBC are urgently needed. Cholesterol is an essential structural and functional component of cell membranes that is necessary for the growth of both primary tumors and metastatic colonies that reside in distant organs. Therefore, inhibiting cholesterol production is an attractive therapeutic strategy. Treatment with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently, we targeted oxidosqualene cyclase (OSC), an enzyme that occurs downstream of HMGCoA-reductase in the cholesterol biosynthetic pathway, to disrupt tumor metastasis. Potent small molecule inhibitors of OSC have been identified. RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating several forms of primary tumors. We developed a mouse model for studying lung metastasis using a MDA-MB-231 variant (LM2) TNBC cell line obtained from Dr. Massagué (Memorial Sloan-Kettering Cancer Center). These are aggressive cells, and injection of only 2 X 10[5] cells produces detectable lung metastatic colonies in approximately 4-6 weeks. We inoculated female nu/nu mice, 5-6 weeks old, with 2 X 10[5] MDA-MB-231/LM2 cells in 0.1 ml DMEM/F12 medium without serum via tail vein. Five days after tumor cell inoculation animals received 20 mg/kg (n=6), 40 mg/kg (n=7) RO (ip) or vehicle alone (n=7) every other day for two weeks. Animals were then sacrificed, and both lungs harvested and immediately fixed in Bouin’s fixative. Metastatic colonies on the lung surface were identified and counted under a dissecting microscope. Our study showed that RO effectively reduced lung metastasis without affecting animal weight. Compared with 56 + 7 colonies (Mean ± SEM) in controls given vehicle alone, RO significantly reduced lung metastatic colonies to 28 ± 5 in animals exposed to 20 mg/kg and 6 ± 1 in 40 mg/kg treated animals (p<0.05, ANOVA). Reduction in number of colonies in 40 mg/kg group was also significantly different from colonies counted in 20 mg/kg treated animals. Our study is the first to demonstrate that disruption of cholesterol biosynthesis in a model of human TNBC leads to reduced metastasis in lungs. Further studies will determine mechanisms through which RO suppresses TNBC metastasis to the lungs. Supported by Zalk Missouri Professor Endowment Funds. Presentation: 6/3/2024

Genome-wide identification of oxidosqualene cyclase genes regulating natural rubber in Taraxacum kok-saghyz.

Nine TkOSC genes have been identified by genome-wide screening. Among them, TkOSC4-6 might be more crucial for natural rubber biosynthesis in Taraxacum kok-saghyz roots. Taraxacum kok-saghyz Rodin (TKS) roots contain large amounts of natural rubber, inulin, and valuable metabolites. Oxidosqualene cyclase (OSC) is a key member for regulating natural rubber biosynthesis (NRB) via the triterpenoid biosynthesis pathway. To explore the functions of OSC on natural rubber producing in TKS, its gene family members were identified in TKS genome via genome-wide screening. Nine TkOSCs were identified, which were mainly distributed in the cytoplasm. Their family genes experienced a neutral selection during the evolution process. Overall sequence homology analysis OSC proteins revealed 80.23% similarity, indicating a highly degree of conservation. Pairwise comparisons showed a multiple sequence similarity ranging from 57% to 100%. Protein interaction prediction revealed that TkOSCs may interact with baruol synthase, sterol 1,4-demethylase, lupeol synthase and squalene epoxidase. Phylogenetic analysis showed that OSC family proteins belong to two branches. TkOSC promoter regions contain cis-acting elements related to plant growth, stress response, hormones response and light response. Protein accumulation analysis demonstrated that TkOSC4, TkOSC5 and TkOSC6 proteins had strong expression levels in the root, latex and plumular axis. Comparison of gene expression patterns showed TkOSC1, TkOSC4, TkOSC5, TkOSC6, TkOSC7, TkOSC8 and TkOSC9 might be important in regulating NRB. Combination of gene and protein results revealed TkOSC4-6 might be more crucial, and the data might contribute to a more profound understanding of the roles of OSCs for NRB in TKS roots.

Genomic insights into ecological adaptation of oaks revealed by phylogenomic analysis of multiple species

Understanding the ecological adaptation of tree species can not only reveal the evolutionary potential but also benefit biodiversity conservation under global climate change. Quercus is a keystone genus in Northern Hemisphere forests, and its wide distribution in diverse ecosystems and long evolutionary history make it an ideal model for studying the genomic basis of ecological adaptations. Here we used a newly sequenced genome of Quercus gilva, an evergreen oak species from East Asia, with 18 published Fagales genomes to determine how Fagaceae genomes have evolved, identify genomic footprints of ecological adaptability in oaks in general, as well as between evergreen and deciduous oaks. We found that oak species exhibited a higher degree of genomic conservation and stability, as indicated by the absence of large-scale chromosomal structural variations or additional whole-genome duplication events. In addition, we identified expansion and tandem repetitions within gene families that contribute to plant physical and chemical defense (e.g., cuticle biosynthesis and oxidosqualene cyclase genes), which may represent the foundation for the ecological adaptation of oak species. Circadian rhythm and hormone-related genes may regulate the habits of evergreen and deciduous oaks. This study provides a comprehensive perspective on the ecological adaptations of tree species based on phylogenetic, genome evolutionary, and functional genomic analyses.

Cloning and functional characterization of oxidosqualene cyclase gene for α-amyrin synthesis

Ursolic acid has gradually attracted much attention due to its unique pharmacological activities and valuable market value in recent years. Currently, ursolic acid is mostly extracted from loquat leaves, but the plant extraction method has low yield and high cost, and chemical synthesis is not readily available, so the biosynthesis method provides a new source for ursolic acid. α-amyrin acts as the main precursor for the synthesis of ursolic acid, and its yield is positively correlated with ursolic acid yield. Oxidosqualene cyclase(OSC) belongs to a multigene family which can catalyze the common precursor 2,3-oxidosqualene to generate different types of triterpene backbones, and plays a decisive role in the synthesis of triterpenoids. However, there are fewer reported key genes catalyzing the synthesis of α-amyrin in medicinal plants, and the yield and proportion of α-amyrin in the catalyzed products have always been a focus of research. In this study, ItOSC2, MdOSC1, AaOSC2 and CrAS, four enzymes capable of catalyzing the production of α-amyrin from 2,3-oxidosqualene, were cloned from Iris tectorum, Malus domestica, Artemisia annua and Catharanthus roseus, subject to sequence alignment and phylogenetic tree analyses, and transformed into Saccharomyces cerevisiae as plasmids. After 7 days of fermentation, the yield and proportions of α-amyrin, β-amyrin and ergosterol were measured. Finally, AaOSC2 with the best ability to catalyze the generation of α-amyrin was filtered out, providing a key gene element for the later construction of engineered yeast strains with high production of α-amyrin and ursolic acid.

Adjusting Catalytic Activity of β-Amyrin Synthase GgBAS by Utilizing the Plasticity Residues of an Active Site.

β-Amyrin synthase (bAS) is a representative plant oxidosqualene cyclase (OSC), and previous studies have identified many functional residues and mutants that can alter its catalytic activity. However, the regulatory mechanism of the active site architecture for adjusting the catalytic activity remains unclear. In this study, we investigate the function of key residues and their regulatory effects on the catalytic activity of Glycyrrhiza glabra β-amyrin synthase (GgbAS) through molecular dynamics simulations and site-directed mutagenesis experiments. We identified the plasticity residues located in two active site regions and explored the interactions between these residues and tetracyclic/pentacyclic intermediates. Based on computational and experimental results, we further categorize these plasticity residues into three types: effector, adjuster, and supporter residues, according to their functions in the catalytic process. This study provides valuable insights into the catalytic mechanism and active site plasticity of GgbAS, offering important references for the rational enzyme engineering of other OSC enzyme.

Comparative transcriptome and metabolite profiling reveal diverse pattern of CYP-TS gene expression during corosolic acid biosynthesis in Lagerstroemia speciosa (L.) Pers.

Extensive leaf transcriptome profiling and differential gene expression analysis of field grown and elicited shoot cultures of L. speciosa suggest that differential synthesis of CRA is mediated primarily by CYP and TS genes, showing functional diversity. Lagerstroemia speciosa L. is a tree species with medicinal and horticultural attributes. The pentacyclic triterpene, Corosolic acid (CRA) obtained from this species is widely used for the management of diabetes mellitus in traditional medicine. The high mercantile value of the compound and limited availability of innate resources entail exploration of alternative sources for CRA production. Metabolic pathway engineering for enhanced bioproduction of plant secondary metabolites is an attractive proposition for which, candidate genes in the pathway need to be identified and characterized. Therefore, in the present investigation, we focused on the identification of cytochrome P450 (CYP450) and oxidosqualene cyclases (OSC) genes and their differential expression during biosynthesis of CRA. The pattern of differential expression of these genes in the shoot cultures of L. speciosa, elicited with different epigenetic modifiers (azacytidine (AzaC), sodium butyrate (NaBu) and anacardic acid (AA)), was studied in comparison with field grown plant. Further, in vitro cultures with varying (low to high) concentrations of CRA were systematically assessed for the expression of CYP-TS and associated genes involved in CRA biosynthesis by transcriptome sequencing. The sequenced samples were de novo assembled into 180,290 transcripts of which, 92,983 transcripts were further annotated by UniProt. The results are collectively given in co-occurrence heat maps to identify the differentially expressed genes. The combined transcript and metabolite profiles along with RT-qPCR analysis resulted in the identification of CYP-TS genes with high sequence variation. Further, instances of concordant/discordant relation between CRA biosynthesis and CYP-TS gene expression were observed, indicating functional diversity in genes.

Minor Hydroxylated Triterpenoids Produced in Engineered Yeast by the Enzymes OSC and CYP716s from the Plant Enkianthus chinensis and Their Anti-Inflammatory and Hepatoprotective Activities.

Triterpenoids are a type of specialized metabolites that exhibit a wide range of biological activities. However, the availability of some minor triterpenoids in nature is limited, which has hindered our understanding of their pharmacological potential. To overcome this limitation, heterologous biosynthesis of triterpenoids in yeast has emerged as a promising and time-efficient production platform for obtaining these minor compounds. In this study, we analyzed the transcriptomic data of Enkianthus chinensis to identify one oxidosqualene cyclase (EcOSC) gene and four CYP716s. Through heterologous expression of these genes in yeast, nine natural pentacyclic triterpenoids, including three skeleton products (1-3) produced by one multifunctional OSC and six minor oxidation products (4-9) catalyzed by CYP716s, were obtained. Of note, we discovered that CYP716E60 could oxidize ursane-type and oleanane-type triterpenoids to produce 6β-OH derivatives, marking the first confirmed C-6β hydroxylation in an ursuane-type triterpenoid. Compound 9 showed moderate inhibitory activity against NO production and dose-dependently reduced IL-1β and IL-6 production at the transcriptional and protein levels. Compounds 1, 2, 8, and 9 exhibited moderate hepatoprotective activity with the survival rates of HepG2 cells from 61% to 68% at 10 μM.

Promiscuous Oxidosqualene Cyclases from Neoalsomitra integrifoliola Catalyzing the Formation of Tetracyclic, Pentacyclic, and Heterocyclic Triterpenes.

Six oxidosqualene cyclases (NiOSC1-NiOSC6) from Neoalsomitra integrifoliola were characterized for the biosynthesis of diverse triterpene scaffolds, including tetracyclic and pentacyclic triterpenes from the 2,3-oxidosqualene (1) and oxacyclic triterpenes from the 2,3:22,23-dioxidosqualene (2). NiOSC1 showed high efficiency in the production of naturally rare (20R)-epimers of oxacyclic triterpenes. Mutagenesis results revealed that the NiOSC1-F731G mutant significantly increased the yields of (20R)-epimers compared to the wild type. Homology modeling and molecular docking elucidated the origin of the (20R)-configuration in the epoxide addition step.

Functional characterization of genes related to triterpene and flavonoid biosynthesis in Cyclocarya paliurus.

The oxidosqualene cyclases (OSCs) generating triterpenoid skeletons in Cyclocarya paliurus were identified for the first time, and two uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyzing the glycosylation of flavonoids were characterized. Cyclocarya paliurus, a native rare dicotyledonous plant in China, contains an abundance of triterpenoid saponins and flavonoid glycosides that exhibit valuable pharmaceutical effects in preventing hypertension, hyperlipidemia, and diabetes. However, the molecular mechanism explaining the biosynthesis of triterpenoid saponin and flavonoid glycoside in C. paliurus remains unclear. In this study, the triterpene content in different tissues and the expression pattern of genes encoding the key enzymes associated with triterpenoid saponin and flavonoid glycoside biosynthesis were studied using transcriptome and metabolome analysis. The eight upstream oxidosqualene cyclases (OSCs) involved in triterpenoid saponin biosynthesis were functionally characterized, among them CpalOSC6 catalyzed 2,3;22,23-dioxidosqualene to form 3-epicabraleadiol; CpalOSC8 cyclized 2,3-oxidosqualene to generate dammarenediol-II; CpalOSC2 and CpalOSC3 produced β-amyrin and CpalOSC4 produced cycloartenol, while CpalOSC2-CpalOSC5, CpalOSC7, and CpalOSC8 all produced lanosterol. However, no catalytic product was detected for CpalOSC1. Moreover, two downstream flavonoid uridine diphosphate (UDP)-glycosyltransferases (UGTs) (CpalUGT015 and CpalUGT100) that catalyze the last step of flavonoid glycoside biosynthesis were functionally elucidated. These results uncovered the key genes involved in the biosynthesis of triterpenoid saponins and flavonoid glycosides in C. paliurus that could be applied to produce flavonoid glycosides and key triterpenoid saponins in the future via a synthetic strategy.

Aquilaria sinensis: An Upstart Resource for Cucurbitacin Production Offers Insights into the Origin of Plant Bitter (Bi) Gene Clusters.

Cucurbitacins, oxygenated tetracyclic triterpenoids that are found mainly in the Cucurbitaceae family, play essential roles as defensive compounds, serving as allomones against herbivores and pathogens and as signals for insect-parasite recognition. These compounds also exhibit various pharmacological effects. The biosynthesis of cucurbitacins is largely regulated by the bitter (Bi) gene, encoding an oxidosqualene cyclase, which catalyzes the conversion of 2,3-oxidosqualene into cucurbitadienol, a common precursor for cucurbitacin synthesis. Previous studies focused on uncovering the Bi gene clusters in Cucurbitaceae, but their presence in other cucurbitacin-producing plants remained unexplored. Here, the evolutionary history of Bi genes and their clusters were investigated in twenty-one plant genomes spanning three families based on chemotaxonomy. Nineteen Bi genes were identified in fourteen Cucurbitaceae, four Begoniaceae, and one Aquilaria species. Phylogenetic analysis suggested that the genome of Aquilaria sinensis contained the earliest Bi gene clusters in this dataset. Moreover, the genomic analysis revealed a conserved microsynteny of pivotal genes for cucurbitacin biosynthesis in Cucurbitaceae, while interspersed Bi gene clusters were observed in Begoniaceae, indicating rearrangements during plant Bi gene cluster formation. The bitter gene in A. sinensis was found to promote cucurbitadienol biosynthesis in the leaves of Nicotiana benthamiana. This comprehensive exploration of plant Bi genes and their clusters provides valuable insights into the genetic and evolutionary underpinnings of cucurbitacin biosynthesis. These findings offer prospects for a deeper understanding of cucurbitacin production and potential genetic resources for their enhancement in various plants.

Identification and Functional Characterization of Oxidosqualene Cyclases from Medicinal Plant Hoodia gordonii.

Oxidosqualene cyclases (OSCs) are the key enzymes accountable for the cyclization of 2,3-oxidosqualene to varied triterpenoids and phytosterols. Hoodia gordonii (from the family Apocynaceae), a native of the Kalahari deserts of South Africa, Namibia, and Botswana, is being sold as a prevalent herbal supplement for weight loss. The appetite suppressant properties are attributed to P57AS3, an oxypregnane steroidal glycoside. At the molecular level, the enzymes involved in the biosynthesis of triterpenes and phytosterols from H. gordonii have not been previously reported. In the current study, predicted transcripts potentially encoding oxidosqualene cyclases were recognized first by searching publicly available H. gordonii RNA-seq datasets. Two OSC-like sequences were selected for functional analysis. A monofunctional OSC, designated HgOSC1 which encodes lupeol synthase, and HgOSC2, a multifunctional cycloartenol synthase forming cycloartenol and other products, were observed through recombinant enzyme studies. These studies revealed that distinct OSCs exist for triterpene formation in H. gordonii and provided opportunities for the metabolic engineering of specific precursors in producing phytosterols in this plant species.

OXIDOSQUALENE CYCLASE 1 and 2 influence triterpene biosynthesis and defense in Nicotiana attenuata.

Triterpenes are a class of bioactive compounds with diverse biological functions, playing pivotal roles in plant defense against biotic stressors. Oxidosqualene cyclases (OSCs) serve as gatekeepers in the biosynthesis of triterpenes. In this study, we utilized a Nicotiana benthamiana heterologous expression system to characterize NaOSC1 from Nicotiana attenuata as a multifunctional enzyme capable of synthesizing lupeol, dammarenediol II, 3-alpha,20-lupanediol, and 7 other triterpene scaffolds. We also demonstrated that NaOSC2 is, in contrast, a selective enzyme, producing only the β-amyrin scaffold. Through virus-induced gene silencing and in vitro toxicity assays, we elucidated the roles of NaOSC1 and NaOSC2 in the defense of N. attenuata against Manduca sexta larvae. Metabolomic and feature-based molecular network analyses of leaves with silenced NaOSC1 and NaOSC2 unveiled 3 potential triterpene glycoside metabolite clusters. Interestingly, features identified as triterpenes within these clusters displayed a significant negative correlation with larval mass. Our study highlights the pivotal roles of NaOSC1 and NaOSC2 from N. attenuata in the initial steps of triterpene biosynthesis, subsequently influencing defense against M. sexta through the modulation of downstream triterpene glycoside compounds.

Characterization of oxidosqualene cyclases involved in pentacyclic triterpene biosynthesis in Korean chestnut (Castanea crenata)

Characterization of oxidosqualene cyclases involved in pentacyclic triterpene biosynthesis in Korean chestnut (Castanea crenata)

Structural and Catalytic Insight into the Unique Pentacyclic Triterpene Synthase TwOSC.

The oxidosqualene cyclase (OSC) catalyzed cyclization of the linear substrate (3S)-2,3-oxidosqualene to form diverse pentacyclic triterpenoid (PT) skeletons is one of the most complex reactions in nature. Friedelin has a unique PT skeleton involving a fascinating nine-step cation shuttle run (CSR) cascade rearrangement reaction, in which the carbocation formed at C2 moves to the other side of the skeleton, runs back to C3 to yield a friedelin cation, which is finally deprotonated. However, as crystal structure data of plant OSCs are lacking, it remains unknown why the CSR cascade reactions occur in friedelin biosynthesis, as does the exact catalytic mechanism of the CSR. In this study, we determined the first cryogenic electron microscopy structure of a plant OSC, friedelin synthase, from Tripterygium wilfordii Hook. f (TwOSC). We also performed quantum mechanics/molecular mechanics simulations to reveal the energy profile for the CSR cascade reaction and identify key residues crucial for PT skeleton formation. Furthermore, we semirationally designed two TwOSC mutants, which significantly improved the yields of friedelin and β-amyrin, respectively.

Structural and Catalytic Insight into the Unique Pentacyclic Triterpene Synthase TwOSC

AbstractThe oxidosqualene cyclase (OSC) catalyzed cyclization of the linear substrate (3S)‐2,3‐oxidosqualene to form diverse pentacyclic triterpenoid (PT) skeletons is one of the most complex reactions in nature. Friedelin has a unique PT skeleton involving a fascinating nine‐step cation shuttle run (CSR) cascade rearrangement reaction, in which the carbocation formed at C2 moves to the other side of the skeleton, runs back to C3 to yield a friedelin cation, which is finally deprotonated. However, as crystal structure data of plant OSCs are lacking, it remains unknown why the CSR cascade reactions occur in friedelin biosynthesis, as does the exact catalytic mechanism of the CSR. In this study, we determined the first cryogenic electron microscopy structure of a plant OSC, friedelin synthase, from Tripterygium wilfordii Hook. f (TwOSC). We also performed quantum mechanics/molecular mechanics simulations to reveal the energy profile for the CSR cascade reaction and identify key residues crucial for PT skeleton formation. Furthermore, we semirationally designed two TwOSC mutants, which significantly improved the yields of friedelin and β‐amyrin, respectively.

THU542 Inhibiting Oxidosqualene Cyclase Suppresses Cancer Stem-like Cells And Growth Of Epithelial Ovarian Cancer Cells In Vitro And In Vivo

Abstract Disclosure: Y. Liang: None. K.P. Nephew: None. S.M. Hyder: None. Epithelial ovarian cancer (EOC) is an aggressive and deadly disease and despite concerted efforts to develop new strategies for preventing and treating the disease, almost 25,000 new cases are reported each year in the United States. Unfortunately, patients with ovarian cancer have poor prognosis due to the emergence of drug resistant cells and subsequent metastasis and have the highest mortality rate of all known gynecological malignancies. Standard treatments include systemic high-dose toxic chemotherapeutic drugs, but drug-resistance almost always occurs. Consequently, novel and more effective non-toxic therapies for ovarian cancer are urgently needed. The biosynthetic pathway leading to cholesterol production is an attractive therapeutic target since cholesterol is an essential structural and functional component of cell membranes and is necessary for tumor growth. Treatment with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently, alternative approaches to inhibit cholesterol biosynthesis are being considered. Epithelial ovarian cancer cells express enzymes in the cholesterol biosynthetic pathway. With this is mind we inhibited a key enzyme in the pathway, oxidosqualene cyclase (OSC), with a view to disrupting cholesterol biosynthesis and disrupting the progression of ovarian cancer. Potent small molecule inhibitors of OSC have been identified, and RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating breast and prostate cancer. Since OSC acts downstream of HMG-CoA reductase during cholesterol biosynthesis, its inhibition is not likely to be associated with the adverse effects caused by statins. We found in short-term assays (24-48 h) that pharmacological doses very effectively reduced the viability of ovarian tumor cells (SK-OV-3 and OVCAR-3), determined by Sulforhodamine B colorimetric assay. In long-term assays (7 days), nM concentrations of the drug were also effective. Importantly, RO (20-40 mg/kg/day; ip) significantly suppressed tumor xenografts derived from ovarian cancer cells (SK-OV-3; 336 + 60 mm3 vs 171 + 20 mm3) with no toxicity to experimental animals (27 treatments once tumors reached 100 mm3). Mechanistically, RO induced apoptosis in EOC, and suppressed ALDH activity, indicating that the inhibitor suppresses stem cell-like activity of EOC. Our study is the first to demonstrate disruption of cholesterol biosynthesis in human ovarian cancer cells as a novel and potent means by which to suppress their growth. Further studies will determine whether combining RO with chemotherapeutic drugs is an even more effective therapeutic approach to EOC, and examine the effects of RO on combating the development of drug-resistant ovarian cancer. Presentation: Thursday, June 15, 2023