Farnesyl Diphosphate Research Articles

Unravelling the aromatic symphony: redirecting bifunctional mushroom synthases towards linalool monofunctionality

Enzymes are the cornerstone of biocatalysis, biosynthesis and synthetic biology. However, their applicability is often limited by low substrate selectivity. A prime example is the bifunctional linalool/nerolidol synthase (LNS) that can use both geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) to produce linalool and nerolidol, respectively. This bifunctionality can lead to undesired byproducts in synthetic biology applications. To enhance enzyme specificity and create monofunctional linalool synthases, we modified amino acids in the loop between helices C and D of four bifunctional mushroom LNSs. Through these modifications, we successfully shifted the substrate preference of two LNSs (ApLNS from Agrocybe pediades and HsLNS from Hypholoma sublateritium) from FPP towards GPP. Although complete monofunctionality was not achieved, we significantly increased linalool yield by 13 times while minimizing nerolidol production to 1% of the wildtype HsLNS. Docking simulations revealed a substantial reduction in the FPP binding score compared to that of the wildtype. Molecular dynamics simulations suggested that Tyr300 in the apo HsLNS mutant has a greater tendency to adopt an inward orientation. Together with Met77, the inward-facing Tyr300 creates a steric barrier that prevents the longer FPP molecule from entering the substrate binding pocket, thereby greatly reducing its activity towards FPP. This study demonstrates the potential of enzyme engineering to design substrate-specific terpene synthases, which is a challenging task and few successful examples are available. The insights gained can inform future enzyme design efforts, including the development of artificial intelligence models.

Mechanism of sodium nitroprusside regulating ginseng quality

The roots of Panax ginseng C. A. Meyer (ginseng) are one of the traditional medicinal herbs in Asian countries and is known as the “king of all herbs”. The most important active components of ginseng are the secondary metabolite saponins, which are closely related to ecological stress. Unsuitable ecological stress can generate a large amount of reactive oxygen species (ROS), by which the secondary metabolism is regulated, and the quality of herbs can be significantly improved. The purpose of this study was to investigate the effect of sodium nitroprusside (SNP) treatment on the quality of fresh ginseng roots. In this study, 5-year-old fresh ginseng was exposed to 0.1, 0.5, and 2 mmol/L SNP, a nitric oxide (NO) donor for five consecutive days. SNP significantly increased the levels of O2·−, H2O2, malondialdehyde (MDA), NADPH oxidase (NOX), superoxide dismutase (SOD), catalase (CAT), peroxides (POD), ascorbate peroxidase (APX), glutathione reductase (GR), ascorbate (AsA) and GSH/GSSG. The main root treated by 0.5 mmol/L SNP for three days was the best, with the activities of the key enzymes of the ginsenoside synthesis pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), farnesyl pyrophosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), and dammarane diol-II synthase (DS) activities increased markedly; the ginsenosides Rg1 + Re, Rb1, Rf, Rc, Rg2 + Rh1 and the total ginsenoside contents increased by 51.0%, 77.7%, 44.6%, 26.8%, 63.2% and 48.2%, respectively, but only a trace amount of the ginsenoside monomer Rb2 decreased 23.4%. The fibrous roots treated by 0.1 mmol/L SNP for four days showed the best effect, HMGCR, FPS, SS, SE, and DS also increased significantly; ginsenosides Rg1 + Re, Rb1, Ro, Rc, Rf, Rb3, Rb2, and total saponin contents increased 37.6%, 47.8%, 34.2%, 75.1%, 51.0%, 49.4%, 28.3%, and 20.4%, respectively. The 1,3-diphosphoglycerate (1,3-DPG) and phosphoenolpyruvate carboxylase (PEPC), related to primary metabolism, were also significantly elevated. The Morris water maze (MWM), histopathological analysis and oxidative stress indexes in brain tissues were used to evaluate the anti-aging effect, indicating that the SNP-treated ginseng further ameliorated D-gal-induced the impaired memory function and oxidative stress in mice, implying the efficacy of SNP-treated ginseng was better than untreated ginseng’s. SNP can build the physiological state of ginseng under ecological stress, stimulate the antioxidant protection mechanism, increase the secondary metabolites, and improve the quality of ginseng.

Chemoenzymatic Formation of Oxa-Terpenoids by Sesqui- and Diterpene Synthase-Mediated Biotransformations with 9-Oxy-FPP Ether Derivatives.

Farnesyl pyrophosphate derivatives bearing an additional oxygen atom at position 5 proved to be very suitable for expanding the substrate promiscuity of sesquiterpene synthases (STSs) and the formation of new oxygenated terpenoids. Insertion of an oxygen atom in position 9, however, caused larger restraints that led to restricted acceptance by STSs. In order to reduce some of the proposed restrictions, two FPP-ether derivatives with altered substitution pattern around the terminal olefinic double bond were designed. These showed improved promiscuity toward different STSs. Four new cyclized terpenoids with an embedded ether group were isolated and characterized. In the case of two cyclic enol ethers, also the corresponding "hydrolysis" products, linear hydroxyaldehydes, were isolated. Interestingly, all cyclization products originate from an initial 1 → 12 cyclization unprecedented when native farnesyl pyrophosphate serves as a substrate. We found that the most suitable FPP derivative with an additional oxygen at position 9 does not carry any methyl group on the terminal alkene, which likely reduces steric congestion when the preferred conformation for cyclization is adopted in the active site.

Transcriptomic Analysis of Neocaridina denticulata sinensis Gills Following FPPS Knockdown Reveals Its Regulatory Role in Immune Response.

Farnesyl pyrophosphate synthase (FPPS) is a key enzyme in the terpenoid biosynthesis pathway, responsible for converting isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) into farnesyl pyrophosphate (FPP). In crustaceans, FPPS plays an important role in various physiological processes, particularly in synthesizing the crustacean-specific hormone methyl farnesoate (MF). This study analyzed the evolutionary differences in the physicochemical properties, subcellular localization, gene structure, and motif composition of FPPS in Neocaridina denticulata sinensis (named NdFPPS) compared to other species. The significant evolutionary divergence of FPPS was observed in crustaceans, likely linked to its role in MF synthesis. After the RNA interference (RNAi)-mediated knockdown of NdFPPS, transcriptomic analysis of gills revealed the significant enrichment of differentially expressed genes (DEGs) in pathways related to metabolism and immunity. Gene set enrichment analysis (GSEA) showed that most of these immune-related pathways were significantly suppressed, suggesting that NdFPPS may indirectly regulate the immune response by modulating metabolic levels. During the early stages of Vibrio parahaemolyticus infection, the expression of NdFPPS in the gills was significantly downregulated and subsequently returned to its original levels. Overall, our results provide new perspectives on the role of FPPS in immune regulation and enrich the functional information of FPPS.

Nicht‐Kanonische C16 Homoterpen‐Biosynthese Weitverbreitet in Actinobakterien

AbstractEin neuartiger Biosyntheseweg zur seltenen und wenig Erforschten nicht‐kanonischen Familie der Homoterpene wurde in Actinobakterien durch gezieltes Genome‐Mining und enzymatische In vitro‐Rekonstitution entdeckt. Der Weg umfasst eine anfängliche methylierungsinduzierte Doppelbindungsisomerisierung von Farnesyldiphosphat (FPP) zu (2E,7E)‐6‐Methylfarnesyldiphosphat, katalysiert durch eine neuartige Familie von Methyltransferasen mit dualer Funktion. Das resultierende lineare C16‐Doppelbindungsisomer von FPP stellt das spezifische Substrat für eine neue Familie von Typ‐I‐Terpenzyklasen dar, die diverse Zyklisierungsreaktionen katalysieren. Funktionelle Charakterisierung von neun Enzympaaren führte zur Entdeckung von fünf bisher unbeschriebenen Homoterpen‐Naturstoffen. Die entdeckte Enzymologie ermöglicht die Entwicklung biokatalytischer und genetisch programmierbarer Systeme zur Herstellung methylierter Terpene mit potenziell neuartigen Eigenschaften („Magic‐Methyl‐Effekt“).

Non-Canonical C16 Homoterpene Biosynthesis Widespread in Actinobacteria.

A novel biosynthetic pathway towards the rare and underexplored non-canonical family of homoterpenes was discovered in actinobacteria through targeted genome mining and enzymatic in vitro reconstitution. The pathway comprises initial methylation-induced double bond isomerization of farnesyl diphosphate (FPP) to (2E,7E)-6-methyl-farnesyl diphosphate, catalyzed by a novel family of methyltransferases with unique dual function. The resulting linear C16 double bond isomer of FPP constitutes the specific substrate for a distinct family of type I terpene cyclases, catalyzing diverse cyclization reactions. Functional characterization of nine enzyme pairs led to discovery of five unprecedented homoterpene natural products. The enzymological novelty enables the development of novel biocatalytic and genetically programmable synthetic strategies towards methylated terpenoids with potentially unique properties ("magic methyl effect").

Molecular Insight into the Catalytic Mechanism of the Sesquiterpene Cyclase BcABA3.

BcABA3 is an unusual sesquiterpene synthase that lacks the conserved DDxxD and DTE/NSE motifs. Despite this, it can catalyze the conversion of farnesyl diphosphate to 2Z,4E-α-ionylideneethane. We used structure prediction, multiscale simulations, and site-directed mutagenesis experiments to investigate BcABA3 and its catalytic mechanism. BcABA3 has structural similarity to typical class I terpenoid cyclases in its active site. Based on simulation results, we identified two discontinuous glutamate residues, E124 and E88, which compensate for the absence of the aspartate-rich DDxxD motif. Quantum chemical calculations show that BcABA3 adopts a direct rotation mechanism for allyl cation isomerization rather than via the nerolidyl diphosphate. Then, it can achieve a successive proton transfer reaction, which is difficult to achieve by intramolecular rearrangement via the protruding outward carbonyl oxygen of A206. This reaction is then directed forward by two relatively stable intermediates containing a cation-conjugated double-bond structure. E124 is also proposed as the proton receptor in the final deprotonation to couple this step with 2Z,4E-α-ionylideneethane release. These findings provide valuable insight into the catalytic mechanisms of BcABA3 and can aid in its engineering, which will facilitate studies of abscisic acid biosynthesis.

An Alkyne-Containing Isoprenoid Analogue Based on a Farnesyl Diphosphate Scaffold Is a Biologically Functional Universal Probe for Proteomic Analysis.

Prenylation consists of the modification of proteins with either farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP) at a cysteine near the C-terminus of target proteins to generate thioether-linked lipidated proteins. In recent work, metabolic labeling with alkyne-containing isoprenoid analogues including C15AlkOPP has been used to identify prenylated proteins and track their levels in different diseases. Here, a systematic study of the impact of isoprenoid length on proteins labeled with these probes was performed. Chemical synthesis was used to generate two new analogues, C15hAlkOPP and C20AlkOPP, bringing the total number of compounds to eight used in this study. Enzyme kinetics performed in vitro combined with metabolic labeling in cellulo, resulted in the identification of 8 proteins for C10AlkOPP, 70 proteins for C15AlkOPP, 41 proteins for C15hAlkOPP, and 7 proteins for C20AlkOPP. While C10AlkOPP was the most selective for farnesylated proteins and C20AlkOPP was most selective for geranylgeranylated proteins, the number of proteins identified using those probes was relatively small. In contrast, C15AlkOPP labeled the most proteins including representatives from all classes of prenylated proteins. Functional analysis of these analogues demonstrated that C15AlkOPP was particularly well suited for biological studies since it was efficiently incorporated in cellulo, was able to confer correct plasma membrane localization of H-Ras protein and complement the effects of GGPP depletion in macrophages to yield correct cell polarization and filopodia. Collectively, these results indicate that C15AlkOPP is a biologically functional, universal probe for metabolic labeling experiments that has minimal effects on cellular physiology.

Compartmentalized Sesquiterpenoid Biosynthesis and Functionalization in the Chlamydomonas reinhardtii Plastid.

Terpenoids play key roles in cellular metabolism and can have specialized functions. Their heterologous production in microbial hosts offers an alternative to natural extraction. Here, we developed a subcellular engineering approach in the model green alga Chlamydomonas reinhardtii by targeting both sesquiterpenoid synthases and cytochrome P450s (CYPs) to the plastid, exploiting its photosynthetic electron transport chain to drive CYP-mediated oxidation without reductase partners. Nuclear-encoded sesquiterpenoid synthases were expressed with farnesyl pyrophosphate synthase fusions and targeted to the plastid, while CYPs were modified for soluble localization in the plastid stroma by removing transmembrane domains. The plastid environment supported hydroxylation, epoxidation, and oxidation reactions, with functionalization efficiencies reaching 80 % of accumulated products. Carbon source availability influenced product ratios, revealing metabolic flexibility in the engineered pathways. Overall sesquiterpenoid yields ranged between 250-2500 μg L-1 under screening conditions, establishing proof-of-concept for using plastid biochemistry in complex terpenoid biosynthesis. Living two-phase terpenoid extractions with different perfluorinated solvents revealed variable performances based on sesquiterpenoid functionalization and solvent type. This work demonstrates that photosynthetic electron transport can drive CYP-mediated functionalization in engineered subcellular compartments. However, improvements in photobioreactor cultivation concepts will be required to facilitate the use of algal chassis for scaled production.

Fine-Tuning the Function of Farnesene Synthases for Selective Synthesis of Farnesene Stereoisomers.

Farnesene synthase from Artemisia annua (AaFS) catalyzes the reaction from farnesyl pyrophosphate (FPP) to give the sesquiterpene β-farnesene, a key building block for the biosynthesis of vitamin E. However, an insufficient yield of β-farnesene precludes its industrialization. Understanding the mechanism would be essential for attaining β-farnesene in high yield. Guided by structure-based enzyme engineering, we designed several potent variants, among which L326I increased the β-farnesene yield from 450.65 to 3877.42 mg/L. Furthermore, we found that the function of β-farnesene synthase AaFS can be modulated at two positions; W299 is responsible for tuning the enzyme's function to give its isomeric product α-farnesene and Y402 is the key residue for diverting from the linear farnesene products to the monocyclic α-bisabolol product. These findings provide valuable insights into the catalytic mechanism and functional modulation of farnesene synthases and set the basis for rational engineering of farnesene synthases for selective biosynthesis of diverse sesquiterpene natural products.

Synthetic indole derivatives as an antibacterial agent inhibiting respiratory metabolism of multidrug-resistant gram-positive bacteria

The survival of modern medicine depends heavily on the effective prevention and treatment of bacterial infections, are threatened by antibacterial resistance. The increasing use of antibiotics and lack of stewardship have led to an increase in antibiotic-resistant pathogens, so the growing issue of resistance can be resolved by emphasizing chemically synthesized antibiotics. This study discovered SMJ-2, a synthetic indole derivative, is effective against all multidrug-resistant gram-positive bacteria. SMJ-2 has multiple targets of action, but the primary mechanism inhibits respiratory metabolism and membrane potential disruption. SMJ-2 was discovered to interfere with the mevalonate pathway, ultimately preventing the synthesis of farnesyl diphosphate, a precursor to the antioxidant staphyloxanthin, eventually releasing reactive oxygen species, and leading phagocytic cells to destroy pathogens. Additionally, no discernible biochemical and histopathological alterations were found in the mouse acute toxicity model. This study emphasizes mechanistic insights into SMJ-2 as a potential antibacterial with an unusual method of action.

In vivo and in vitro reconstitution of biosynthesis of N-prenylated phenazines revealing diverse phenazine-modifying enzymes.

Phenazine natural products play various roles such as signal molecules, antibiotics, or electron carriers in their producer strains. Among these products, phenazinomycin and lavanducyanin, which are produced by Streptomyces species, are characterized by an N-alkyl modification. Herein, we established the biosynthetic pathways for these two phenazine natural products. Gene-disruption experiments and in vitro reconstitution of the phenazine-tailoring pathway revealed the late steps of the biosynthetic pathway of the phenazines. The class II terpene cyclase homolog Pzm1 catalyzes the cyclization reaction of farnesyl diphosphate to form monocyclic farnesyl diphosphate. Additionally, the prenyltransferase homolog PzmP functions as the N-prenyltransferase of 5,10-dihydrophenazine-1-carboxylic acid. The flavin monooxygenase homolog PzmS catalyzes the oxidative decarboxylation of prenylated 5,10-dihydrophenazine-1-carboxylic acid to yield phenazinomycin. This study highlights unprecedented modification enzymes for phenazine natural products.

Mevalonate pathway inhibition reduces bladder cancer metastasis by modulating RhoB protein stability and integrin β1 localization

The progression and outcome of bladder cancer (BLCA) are critically affected by the propensity of tumor metastasis. Our previous study revealed that activation of the mevalonate (MVA) pathway promoted migration of BLCA cells; however, the exact mechanism is unclear. Here we show that elevated expression of MVA pathway enzymes in BLCA cells, correlating with poorer patient prognosis by analyzing single-cell and bulk-transcriptomic datasets. Inhibition of the MVA pathway, either through knockdown of farnesyl diphosphate synthase (FDPS) or using inhibitors such as zoledronic acid or simvastatin, led to a marked reduction in BLCA cell migration. Notably, this effect was reversed by administering geranylgeranyl pyrophosphate (GGPP), not farnesyl pyrophosphate (FPP) or cholesterol, indicating the specificity of geranylgeranylation for cell motility. Moreover, we found that RhoB, a Rho GTPase family member, was identified as a key effector of the impact of the MVA pathway on BLCA metastasis. The post-translational modification of RhoB by GGPP-mediated geranylgeranylation influenced its protein stability through the ubiquitin-proteasome pathway. Additionally, overexpression of RhoB was found to block the membrane translocation of integrin β1 in BLCA cells. In summary, our findings underscore the role of the MVA pathway in BLCA metastasis, providing insights into potential therapeutic targets of this malignancy.

TNF inhibitors target a mevalonate metabolite/TRPM2/calcium signaling axis in neutrophils to dampen vasculitis in Behçet’s disease

TNF inhibitors have been used to treat autoimmune and autoinflammatory diseases. Here we report an unexpected mechanism underlying the therapeutic effects of TNF inhibitors in Behçet’s disease (BD), an autoimmune inflammatory disorder. Using serum metabolomics and peripheral immunocyte transcriptomics, we find that polymorphonuclear neutrophil (PMN) from patients with BD (BD-PMN) has dysregulated mevalonate pathway and subsequently increased farnesyl pyrophosphate (FPP) levels. Mechanistically, FPP induces TRPM2-calcium signaling for neutrophil extracellular trap (NET) and proinflammatory cytokine productions, leading to vascular endothelial inflammation and damage. TNF, but not IL-1β, IL-6, IL-18, or IFN-γ, upregulates TRPM2 expression on BD-PMN, while TNF inhibitors have opposite effects. Results from mice with PMN-specific FPP synthetase or TRPM2 deficiency show reduced experimental vasculitis. Meanwhile, analyses of public datasets correlate increased TRPM2 expressions with the clinical benefits of TNF inhibitors. Our results thus implicate FPP-TRPM2-TNF/NETs feedback loops for inflammation aggravation, and novel insights for TNF inhibitor therapies on BD.

Microbial production of 5-epi-jinkoheremol, a plant-derived antifungal sesquiterpene.

Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. In the present study, we constructed a microbial platform for the high-level production of a sesquiterpene from Catharanthus roseus, 5-epi-jinkoheremol, which exhibits strong fungicidal activity. First, the mevalonate and sterol biosynthesis pathways were optimized in engineered yeast to increase the metabolic flux toward the biosynthesis of the precursor farnesyl pyrophosphate. Then, the transcription factor Hac1- and m6A writer Ime4-based metabolic engineering strategies were implemented in yeast to increase 5-epi-jinkoheremol production further. Next, protein engineering was performed to improve the catalytic activity and enhance the stability of the 5-epi-jinkoheremol synthase TPS18, resulting in the variant TPS18I21P/T414S, with the most improved properties. Finally, the titer of 5-epi-jinkoheremol was elevated to 875.25 mg/L in a carbon source-optimized medium in shake flask cultivation. To the best of our knowledge, this is the first study to construct an efficient microbial cell factory for the sustainable production of this antifungal sesquiterpene.IMPORTANCEBiofungicides represent a new and sustainable tool for the control of crop fungal diseases. However, hindered by the high cost of biofungicide production, their use is not as popular as expected. Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. We previously identified a promising sesquiterpenoid biofungicide, 5-epi-jinkoheremol. Here, we constructed a microbial platform for the high-level production of this chemical. The metabolic engineering of the terpene biosynthetic pathway was firstly employed to increase the metabolic flux toward 5-epi-jinkoheremol production. However, the limited catalytic activity of the key enzyme, TPS18, restricted the further yield of 5-epi-jinkoheremol. By using protein engineering, we improved its catalytic efficiency, and combined with the optimization of regulation factors, the highest production of 5-epi-jinkoheremol was achieved. Our work was useful for the larger-scale efficient production of this antifungal sesquiterpene.

Geranylgeranyl diphosphate synthase inhibition impairs osteoclast differentiation, morphology, and resorptive activity.

Nitrogen bisphosphonates, such as zoledronic acid, target the enzyme farnesyl diphosphate synthase (FDPS) in the isoprenoid biosynthetic pathway (IBP), and are the frontline treatment for osteolytic bone diseases. A strong affinity of these agents for bone limits their distribution out of the skeleton. Geranylgeranyl diphosphate synthase (GGDPS) is directly downstream to FDPS in the IBP and novel GGDPS inhibitors such as RAM2061 have been shown to have key drug-like features including prolonged half-life, metabolic stability, and systemic distribution. Furthermore, RAM2061 exerts anti-neoplastic benefits in mouse models of multiple myeloma and Ewing sarcoma. Therefore, we are interested in determining the potential impact of RAM2061 on osteoclast biology and bone remodeling. Studies utilizing undifferentiated RAW264.7 cells demonstrated that treatment with RAM2061 depletes cells of geranylgeranyl diphosphate, impairs protein geranylgeranylation, and induces markers of the unfolded protein response pathway and apoptosis. Differentiation of RAW264.7 cells to mature osteoclasts is disrupted by RAM2061, resulting in decreased numbers of mature osteoclasts, altered morphology, and decreased tartrate-resistant acid phosphatase activity. Treatment of fully differentiated RAW264.7 cells with RAM2061 led to decreased resorptive activity. Confocal microscopy studies revealed that RAM2061 disrupts Cdc42 localization, inhibiting proper actin ring formation in osteoclasts. No significant impact on bone turnover markers or bone histomorphology was observed following a 3-week treatment of CD-1 mice with RAM2061, although decreased numbers of osteoclasts were observed. Liquid chromatography-tandem mass spectrometry studies confirmed accumulation of RAM2061 in bone from the in vivo studies as well as hydroxyapatite binding in vitro. In conclusion, these studies are the first to demonstrate the anti-osteoclastic activity of GGDPS inhibitor treatment and support future studies exploring the therapeutic benefit of this novel therapy in the setting of pathological bone remodeling.

Farnesyl pyrophosphate potentiates dendritic cell migration in autoimmunity through mitochondrial remodelling.

Cellular metabolism modulates dendritic cell (DC) maturation and activation. Migratory dendritic cells (mig-DCs) travelling from the tissues to draining lymph nodes (dLNs) are critical for instructing adaptive immune responses. However, how lipid metabolites influence mig-DCs in autoimmunity remains elusive. Here, we demonstrate that farnesyl pyrophosphate (FPP), an intermediate of the mevalonate pathway, accumulates in mig-DCs derived from mice with systemic lupus erythematosus (SLE). FPP promotes mig-DC survival and germinal centre responses in the dLNs by coordinating protein geranylgeranylation and mitochondrial remodelling. Mechanistically, FPP-dependent RhoA geranylgeranylation promotes mitochondrial fusion and oxidative respiration through mitochondrial RhoA-MFN interaction, which subsequently facilitates the resolution of endoplasmic reticulum stress in mig-DCs. Simvastatin, a chemical inhibitor of the mevalonate pathway, restores mitochondrial function in mig-DCs and ameliorates systemic pathogenesis in SLE mice. Our study reveals a critical role for FPP in dictating mig-DC survival by reprogramming mitochondrial structure and metabolism, providing new insights into the pathogenesis of DC-dependent autoimmune diseases.

Farnesyl pyrophosphate modulates dendritic cell migration in lupus autoimmunity.

Farnesyl pyrophosphate modulates dendritic cell migration in lupus autoimmunity.

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.

Chromosome-Scale Genome and Transcriptomic Analyses Reveal Differential Regulation of Terpenoid Secondary Metabolites in Hericium coralloides.

Construction of the genome of Hericium coralloides, a species of edible mushroom, and identification of the genes involved in terpenoid biosynthesis can determine the biology and genetics of terpenoids. The present study describes the assembly of a high-quality chromosome-scale genome of H. coralloides using Pacbio HiFi sequencing and Hi-C technology. This genome consisted of 13 chromosomes, a total size of 43.6 Mb, contigs of N50 3.6 Mb, GC content at 54%, and BUSCOs integrity of 96.9%. Genes associated with terpenoid biosynthesis were predicted by KEGG enrichment analysis and homologous alignment. The Her011461 and Her008335 genes, encoding proteins in the terpenoid backbone synthesis pathway, were found to encode geranylgeranyl pyrophosphate and farnesyl diphosphate synthases, key enzymes in the biosynthesis of geranylgeranyl diphosphate, a precursor of several diterpenoids. Her011463 was found to be involved in regulating diterpene cyclase. The Her005433, Her006724, Her010605, and Her010608 genes were found to encode sesquiterpene synthesis. Most of these genes were more highly expressed in dikaryotic mycelia than in the primordium and fruiting bodies, indicating that terpenoids may be more abundant in dikaryotic mycelia. To our knowledge, this study is the first to assemble the H. coralloides genome at the chromosome scale and to identify the genes involved in terpenoid biosynthesis.