Cyclase Research Articles

An evolved artificial radical cyclase enables the construction of bicyclic terpenoid scaffolds via an H-atom transfer pathway.

While natural terpenoid cyclases generate complex terpenoid structures via cationic mechanisms, alternative radical cyclization pathways are underexplored. The metal-catalysed H-atom transfer reaction (M-HAT) offers an attractive means for hydrofunctionalizing olefins, providing access to terpenoid-like structures. Artificial metalloenzymes offer a promising strategy for introducing M-HAT reactivity into a protein scaffold. Here we report our efforts towards engineering an artificial radical cyclase (ARCase), resulting from anchoring a biotinylated [Co(Schiff-base)] cofactor within an engineered chimeric streptavidin. After two rounds of directed evolution, a double mutant catalyses a radical cyclization to afford bicyclic products with a cis-5-6-fused ring structure and up to 97% enantiomeric excess. The involvement of a histidine ligation to the Co cofactor is confirmed by crystallography. A time course experiment reveals a cascade reaction catalysed by the ARCase, combining a radical cyclization with a conjugate reduction. The ARCase exhibits tolerance towards variations in the dienone substrate, highlighting its potential to access terpenoid scaffolds.

Biosynthesis of fungal terpenoids.

Covering: up to August 2023Terpenoids, which are widely distributed in animals, plants, and microorganisms, are a large group of natural products with diverse structures and various biological activities. They have made great contributions to human health as therapeutic agents, such as the anti-cancer drug paclitaxel and anti-malarial agent artemisinin. Accordingly, the biosynthesis of this important class of natural products has been extensively studied, which generally involves two major steps: hydrocarbon skeleton construction by terpenoid cyclases and skeleton modification by tailoring enzymes. Additionally, fungi (Ascomycota and Basidiomycota) serve as an important source for the discovery of terpenoids. With the rapid development of sequencing technology and bioinformatics approaches, genome mining has emerged as one of the most effective strategies to discover novel terpenoids from fungi. To date, numerous terpenoid cyclases, including typical class I and class II terpenoid cyclases as well as emerging UbiA-type terpenoid cyclases, have been identified, together with a variety of tailoring enzymes, including cytochrome P450 enzymes, flavin-dependent monooxygenases, and acyltransferases. In this review, our aim is to comprehensively present all fungal terpenoid cyclases identified up to August 2023, with a focus on newly discovered terpenoid cyclases, especially the emerging UbiA-type terpenoid cyclases, and their related tailoring enzymes from 2015 to August 2023.

In silico analysis of sporulene biosynthesis pathway genes in the members of the class Bacilli.

Sporulene, a pentacyclic triterpenoid, was discovered in Bacillus subtilis and is associated with bacterial endospores. However, the study was not further extended, leaving a trail of questions. One such question is what diversity of sporulenes exists among spore-forming members? Considering the sporulene biosynthesis pathway as a fundamental tool to survey the distribution of this molecule, a genome mining study was conducted. Mining for genes encoding putative proteins of sporulene biosynthesis pathway among the class Bacilli members revealed the presence of hepS, hepT, ytpB, and sqhC genes in the members of the family Bacillaceae, Caryophanaceae, Paenibacillaceae, and Sporolactobacillaceae. However, these genes were completely absent in the members of Staphylococcaceae, Lactobacillaceae, Aerococcaceae, Carnobacteriaceae, and Leuconostocaceae. Unlike other probable pathway related proteins, a conserved amino acid domain of putative terpenoid cyclase (YtpB) appeared deep-rooted among the genus Bacillus members. In-depth analysis showed the constant gene arrangement of hepS, hepT, ytpB, and sqhC genes in these members, there by demonstrating the conserved nature of sporulene biosynthesispathway in themembers of the genus Bacillus. Our study suggests confinement of the sporulene biosynthesis pathway to spore-forming members of the class Bacilli, majorly to the genus Bacillus.

A Versatile Nanoluciferase Reporter Reveals Structural Properties Associated with a Highly Efficient, N-Terminal Legionella pneumophila Type IV Secretion Translocation Signal.

Many Gram-negative pathogens rely on type IV secretion systems (T4SS) for infection. One limitation has been the lack of ideal reporters to identify T4SS translocated effectors and study T4SS function. Most reporter systems make use of fusions to reporter proteins, in particular, β-lactamase (TEM) and calmodulin-dependent adenylate cyclase (CYA), that allow detection of translocated enzymatic activity inside host cells. However, both systems require costly reagents and use complex, multistep procedures for loading host cells with substrate (TEM) or for analysis (CYA). Therefore, we have developed and characterized a novel reporter system using nanoluciferase (NLuc) fusions to address these limitations. Serendipitously, we discovered that Nluc itself is efficiently translocated by Legionella pneumophila T4SS in an IcmSW chaperone-dependent manner via an N-terminal translocation signal. Extensive mutagenesis in the NLuc N terminus suggested the importance of an α-helical domain spanning D5 to V9, as mutations predicted to disrupt this structure, with one exception, were translocation defective. Notably, NLuc was capable of translocating several proteins examined when fused to the N or C terminus, while maintaining robust luciferase activity. In particular, it delivered the split GFP11 fragment into J774 macrophages transfected with GFPopt, thereby resulting in in vivo assembly of superfolder green fluorescent protein (GFP). This provided a bifunctional assay in which translocation could be assayed by fluorescence microplate, confocal microscopy, and/or luciferase assays. We further identified an optimal NLuc substrate which allowed a robust, inexpensive, one-step, high-throughput screening assay to identify T4SS translocation substrates and inhibitors. Taken together, these results indicate that NLuc provides both new insight into and also tools for studying T4SS biology. IMPORTANCE Type IV secretion systems (T4SS) are used by Gram-negative pathogens to coopt host cell function. However, the translocation signals recognized by T4SS are not fully explained by primary amino acid sequence, suggesting yet-to-be-defined contributions of secondary and tertiary structure. Here, we unexpectedly identified nanoluciferase (NLuc) as an efficient IcmSW-dependent translocated T4SS substrate, and we provide extensive mutagenesis data suggesting that the first N-terminal, alpha-helix domain is a critical translocation recognition motif. Notably, most existing reporter systems for studying translocated proteins make use of fusions to reporters to permit detection of translocated enzymatic activity inside the host cell. However, existing systems require extremely costly substrates, complex technical procedures to isolate eukaryotic cytoplasm for analysis, and/or are insensitive. Importantly, we found that NLuc provides a powerful, cost-effective new tool to address these limitations and facilitate high-throughput exploration of secretion system biology.

Efficient Bioproduction of Indigo and Indirubin by Optimizing a Novel Terpenoid Cyclase XiaI in Escherichia coli.

Blue indigo dye, an important natural colorant, is used for textiles and food additives worldwide, while another red isomer, indirubin, is the major active ingredient of a traditional Chinese medicine named “Danggui Longhui Wan” for treating various diseases including granulocytic leukemia, cancer, and Alzheimer’s disease. In this work, we constructed a new and highly efficient indigoid production system by optimizing a novel terpenoid cyclase, XiaI, from the xiamycin biosynthetic pathway. Through introducing the flavin-reducing enzyme Fre, tryptophan-lysing and -importing enzymes TnaA and TnaB, and H2O2-degrading enzyme KatE and optimizing the fermentation parameters including temperature, the concentration of isopropyl-β-d-thiogalactopyranoside, and feeding of the l-tryptophan precursor, the final maximum productivity of indigoids by the recombinant strain Escherichia coli BL21(DE3) (XiaI-Fre-TnaAB-KatE) was apparently improved to 101.9 mg/L, an approximately 60-fold improvement to that of the starting strain E. coli BL21(DE3) (XiaI) (1.7 mg/L). In addition, when the fermentation system was enlarged to 1 L in the flask (feeding with 5 mM tryptophan and 10 mM 2-hydroxyindole), the indigoid productivity further increased to 276.7 mg/L at 48 h, including an indigo productivity of 26.0 mg/L and an indirubin productivity of 250.7 mg/L, which has been the highest productivity of indirubin so far. This work provided a basis for the commercial production of bio-indigo and the clinical drug indirubin in the future.

The MLK-1/SCD-4 Mixed Lineage Kinase/MAP3K functions to promote dauer formation upstream of DAF-2/InsR.

The C. elegans dauer is an alternative third stage larva induced by dense population and adverse environmental conditions. Genes whose mutants caused dauer formation constitutive (Daf-c) and dauer formation defective (Daf-d) phenotypes were ordered via epistasis into a signaling network, with upstream DAF-7/TGF-beta and DAF-11/receptor guanylyl cyclase defining sensory branches and downstream DAF-2/Insulin receptor and DAF-12/nuclear hormone receptor executing the dauer decision. Mutations in the Scd genes were defined as incompletely penetrant suppressors of the constitutive dauer phenotype conferred by mutation of the DAF-7/TGF-beta signaling axis. SCD-2 was previously shown to be an ortholog of mammalian ALK (Anaplastic Lymphoma Kinase), a receptor tyrosine kinase. Mutations disrupting the HEN-1/Jeb ligand, SOC-1/DOS/GAB adaptor protein and SMA-5/ERK5 atypical MAP Kinase caused Scd phenotypes similar to that of mutant SCD-2. This group regulated expression from a TGF-beta-responsive GFP reporter. Here we find that a strain harboring a mutation in the uncharacterized SCD-4 is mutant for MLK-1, the C. elegans ortholog of mammalian Mixed Lineage Kinase and Drosophila slipper (slpr), a MAP3 kinase. We validated this finding by showing that a previously characterized deletion in MLK-1 caused a Scd phenotype similar to that of mutant SCD-4 and altered expression from the TGF-beta-responsive GFP reporter, suggesting that SCD-4 and MLK-1 are the same protein. Based on shared phenotypes and molecular identities, we hypothesize that MLK-1 functions as a MAP3K in the SCD-2/ALK cascade that signals through SMA-5/ERK5 MAP Kinase to modulate the output of the TGF-beta cascade controlling dauer formation in response to environmental cues.

Revealing The Motifs, Properties, and Phylogeny of Lupeol Synthase Using Bioinformatics Approach

Lupane-type triterpenoid saponins are potent plants’ secondary metabolites for drug development as they showed various activities such as anticancer, Sarcoplasmic Reticulum Ca 2+ -ATPase (SERCA) activator which is very important for neurons, and antileishmanial. The triterpenoid saponin backbone is produced by cyclization and rearrangement of the 2,3-oxidosqualene precursor by the oxidosqualene cyclase. The type of oxidosqualene cyclase involved determines the type of saponins so it is referred to as a key enzyme. Lupane-type saponins are produced by 2,3-oxidosqualene cyclization through the chair-chair-chair conformation and the formation of various cation intermediates. This study aimed to analyze lupeol synthase, the key enzyme which determines the conversion of 2,3-oxidosqualene into lupane-type saponins. This in silico project was done using bioinformatics programs including Multiple Em for Motif Elicitation (MEME), ProtParam, and Molecular Evolutionary Genetic Analysis (MEGA-X) for relationship analysis. The amino acid sequences analysis using the MEME program showed that lupeol synthase has QW, DCTAE, and CYCR conserved motifs in the oxidosqualene cyclase family even though some evolutions were also present. Analysis of chemical and physical parameters with ProtParam indicated that lupeol synthase had lower stability than lanosterol synthase from Saccharomyces cerevisiae . The phylogenetic tree showed that lupeol synthase was closely related to other plant oxidosqualene cyclases. The results of this study are expected to support the modification strategy determination to increase the production of lupane-type saponins using a biotechnological approach in the pharmaceutical industry.

Structure, catalysis, and inhibition mechanism of prenyltransferase.

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.

Involvement of nNOS, and α1, α2, β1, and β2 Subunits of Soluble Guanylyl Cyclase Genes Expression in Anticonvulsant Effect of Sumatriptan on Pentylenetetrazole-Induced Seizure in Mice.

Epileptic seizure is phenomenon of abnormal synchronous neuronal discharge of a set of neurons in brain as a result of neuronal excitation. Evidence shows the nitric oxide (NO) involvement in neuronal excitability. Moreover, the role of cyclic guanosine monophosphate (cGMP) activation in seizure pathogenesis is well-established. Sumatriptan is a selective agonist of 5-Hydroxytryptamine1B/D auto-receptor, has been reassessed for its neuroprotection. This study was aimed to explore the anticonvulsant effect of sumatriptan through possible involvement of NO-cGMP pathway in mice. For this purpose, the protective effect of sumatriptan on PTZ-induced clonic seizure threshold (CST) was measured using NO-cGMP pathway inhibitors including N(G)-nitro-L-arginine (L-NNA, 1, 5, and 10 mg/kg), 7-nitroindazole (7-NI, 30, 45, and 60 mg/kg), aminoguanidine (AG, 30, 50, and 100 mg/kg), methylene blue (MB, 0.1, 0.5, and 1 mg/kg) and sildenafil (5, 10, and 20 mg/kg). The involvement of nitrergic system was further confirmed by measurement of nitrite levels by Griess reaction. The gene expression of neuronal nitric oxide synthase (nNOS) and subunits of soluble guanylyl cyclase (sGC) was studied using qRT-PCR analysis. Acute administration of sumatriptan (1.2 and 0.3 mg/kg) in combination with subeffective doses of NOS, sGC, and phosphodiesterase 5 inhibitors significantly reversed the PTZ-induced CST (P ≤ 0.001). The nitrite level in prefrontal cortex was significantly attenuated by sumatriptan (P ≤ 0.01). Furthermore, sumatriptan downregulated the PTZ-induced mRNA expression of nNOS (P ≤ 0.01), α1 (P ≤ 0.001), α2 (P ≤ 0.05), and β1 (P ≤ 0.05) genes in cerebral cortex of mice. In conclusion, the anticonvulsant activity of sumatriptan at least, in part, is mediated through inhibiting NO-cGMP pathway.

A Novel ADP-Ribosyl Cyclase (ARC) Regulators

CD38, which is the main NADase enzyme with its ADP-ribose cyclase (ARC) activity, has been proposed to be involved in many human diseases. Therefore, regulating its activity is the main target of this research. This study proposed four novel regulators; nicotinamide, nicotinic acid, gallic acid, and benzamide, these regulators have a remarkable effect on the cyclase activity of ARC. The cyclase activity of ARC was measured using fluorimetric assay, the reaction was initiated with the addition of enzyme to its substrate nicotinamide guanine dinucleotide (NGD) and the production of cyclic GDP-ribose (cGDPR) was measured with different concentrations from proposed activators. The rate of ADP-ribosyl cyclase activity was increased with the addition of low concentrations of Nicotinamide and Nicotinic acid. However, Nicotinamide and Nicotinic acid are known as ARC inhibitors at high concentrations. The increase of ARC activity was also observed with Gallic acid; it is clear that Gallic acid has a strong effect on the activation of ARC and the formation of cGDPR. The last product was Benzamide, these data reflected the activation effect on ARC. Finally, the search for new ADP-ribosyl cyclase activators and the determination of their roles might provide a new therapeutic strategy for several metabolic and inflammatory conditions. How to cite this article: Al-Abady, Z.N., Abdullah Al-Ibadi, I.N., Hatem, O.A., Jabbar, N.K. and Enad, M.A. (2019). A Novel ADP-Ribosyl Cyclase (ARC) Regulators. International Journal of Drug Delivery Technology, 9(4): 640-644. Source of support: Nil. Conflict of interest: None.

Cyclic nucleotide-dependent relaxation in human umbilical vessels.

Umbilical vessels have a low sensitivity to dilate, and this property is speculated to have physiological implications. We aimed to investigate the different relaxing responses of human umbilical arteries (HUAs) and veins (HUVs) to agonists acting through the cAMP and cGMP pathways. Vascular rings were suspended in organ baths for isometric force measurement. Following precontraction with the thromboxane prostanoid (TP) receptor agonist U44069, concentration-response curves to the nitric oxide (NO) donor sodium nitroprusside (SNP), the soluble guanylate cyclase (sGC) stimulator BAY 41-2272, the adenylate cyclase (AC) activator forskolin, the β-adrenergic receptor agonists isoproterenol (ADRB1), salmeterol (ADRB2), and BRL37344 (ADRB3), and the phosphodiesterase (PDE) inhibitors milrinone (PDE3), rolipram (PDE4), and sildenafil (PDE5) were performed. None of the tested drugs induced a relaxation higher than 30% of the U44069-induced tone. Rings from HUAs and HUVs showed a similar relaxation to forskolin, SNP, PDE inhibitors, and ADRB agonists. BAY 41-2272 was significantly more efficient in relaxing veins than arteries. ADRB agonists evoked weak relaxations (< 20%), which were impaired in endothelium-removed vessels or in the presence of the NO synthase inhibitor L-NAME, sGC inhibitor ODQ. PKA and PKG inhibitors impaired ADBR1-mediated relaxation but did not affect ADRB2-mediated relaxation. ADRB3-mediated relaxation was impaired by PKG inhibition in HUAs and by PKA inhibition in HUVs. Although HUA and HUV rings were relaxed by BRL37344, immunohistochemistry and RT-qPCR analysis showed that, compared to ADRB1 and ADRB2, ADRB3 receptors are weakly or not expressed in umbilical vessels. In conclusion, our study confirmed the low relaxing capacity of HUAs and HUVs from term infants. ADRB-induced relaxation is partially mediated by endothelium-derived NO pathway in human umbilical vessels.

Loss of Gpr68 Enhances Hematopoietic Stem Cell Function during Aging

Recent studies implicate metabolic plasticity in fine-tuning hematopoietic stem cell (HSC) homeostasis and function. HSC have been shown to rely on anaerobic glycolysis due to low energetic demand. In contrast, mitochondrial respiration is largely engaged to meet high energetic demand under certain pathophysiological processes, such as differentiation, aging and malignant transformation. The microenvironment, i.e. the HSC niche, has long been known as hypoxic. Glycolytic pathway is not only the metabolic adaptation to the hypoxic niche, but also essential for HSC function at least by providing prosurvival signals. However, glycolysis is reduced in HSC upon aging, which is associated with diminished regeneration ability of HSC. How glycolysis impacts HSC biology is unclear. G protein-coupled receptor 68 (GPR68, also known as OGR1) responds to extracellular acidosis, activating the phospholipase C (PLCb)/calcium pathway or the adenylyl cyclase (CA)/cAMP pathway. The end point product of glycolysis is lactate that leads to extracellular acidosis. This prompts us to examine the role of Gpr68 in HSC biology. Among the hematopoietic stem and progenitor cell (HSPC) subpopulations, we found that Gpr68 expression was most enriched in mouse long-term HSC (LT-HSC, Lin-Sca-1+c-Kit+CD34-CD135-). To understand the function of Gpr68 in HSC biology, we generated a genetic loss of function mouse model (i.e. the Gpr68fl/fl;VavCre+ mice) where Gpr68 was specifically deleted in hematopoietic cells, including the most primitive LT-HSC. We found that the number of LT-HSC was unaltered in bone marrow (BM) from Gpr68fl/fl;VavCre+ mice as compared to wild type (WT) control littermates (i.e. the Gpr68wt/wt;VavCre+ mice), indicating that Gpr68 was not required for HSC homeostasis. To examine the role of Gpr68 in HSC function, we performed competitive bone marrow transplantation (cBMT). The donor-derived chimerism (i.e. the frequency of CD45.2+ cells) was increased in recipients with BM cells from Gpr68fl/fl;VavCre+ mice (12-month old) compared to those from age-matched WT mice after primary cBMT, which was increased even more dramatically after secondary cBMT. This indicates enhanced HSC regeneration ability due to loss of Gpr68. Increased chimerism was observed in CD3+ T lymphocytes, B220+ B lymphocytes and CD11b+ myeloid cells, indicating that both lymphoid and myeloid lineages contributed to enhanced HSC function. Consistently, increased chimerism was also observed in BM HSPC subpopulations except the LT-HSC. Of note, increased chimerism was not observed in recipients that were transplanted with BM cells from young Gpr68fl/fl;VavCre+ mice (2-month old). In addition, BM cells from Gpr68fl/fl;VavCre+ mice produced slightly reduced colonies in methylcellulose, indicating that loss of Gpr68 did not lead to malignant transformation despite enhanced competitiveness of HSC. To further understand the mechanism of Gpr68 in regulating HSC function, we studied cellular features of HSPC subpopulations. The frequency of cells in cell cycle G0 phase was unchanged in LT-HSC from Gpr68fl/fl;VavCre+ mice as compared to WT mice, indicating loss of quiescence may not contribute to enhanced HSC function. Intriguingly, the frequency of Annexin V+ cells was largely reduced in LT-HSC from Gpr68fl/fl;VavCre+ mice as compared to WT mice, suggesting that loss of Gpr68 provides a pro-survival signal in HSC. We next examined two main signaling pathways downstream of Gpr68. Cytosolic calcium levels were reduced in LT-HSC from Gpr68fl/fl;VavCre+ mice as compared to WT mice, indicating Gpr68 may activate a calcium-related proapoptotic pathway. In contrast, intracellular cAMP levels were increased in HSPC, possibly from unknown compensatory mechanism. In addition, Gpr68 expression was reduced in LT-HSC from older mice (12-month old) compared to younger mice (2-month old), indicating reduced Gpr68 function at 12-month of age. This is in line with reduced glycolysis in aged HSC. Our study suggests that Gpr68 mediates a proapoptotic pathway in LT-HSC, providing a negative regulation, possibly at downstream of glycolysis, that limits HSC function. Our study also suggests that inhibiting Gpr68 function would enhance HSC function, especially in aged people or during bone marrow transplantation. Disclosures No relevant conflicts of interest to declare.

Crystal structure of F95Q epi-isozizaene synthase, an engineered sesquiterpene cyclase that generates biofuel precursors β- and γ-curcumene

The saturated hydrocarbon bisabolane is a diesel fuel substitute that can be derived from sesquiterpene precursors bisabolene or curcumene. These sesquiterpenes are generated from farnesyl diphosphate in reactions catalyzed by eponymous terpenoid cyclases, but they can also be generated by engineered terpenoid cyclases in which cyclization cascades have been reprogrammed by mutagenesis. Here, we describe the X-ray crystal structure determination of F95Q epi-isozizaene synthase (EIZS), in which the new activity of curcumene biosynthesis has been introduced and the native activity of epi-isozizaene biosynthesis has been suppressed. F95Q EIZS generates β- and γ-curcumene regioisomers with greater than 50% yield. Structural analysis of the closed active site conformation, stabilized by the binding of 3 Mg2+ ions, inorganic pyrophosphate, and the benzyltriethylammonium cation, reveals a product-like active site contour that serves as the cyclization template. Remolding the active site contour to resemble curcumene instead of epi-isozizaene is the principal determinant of the reprogrammed cyclization cascade. Intriguingly, an ordered water molecule comprises part of the active site contour. This water molecule may also serve as a final proton acceptor, along with inorganic pyrophosphate, in the generation of curcumene regioisomers; it may also contribute to the formation of sesquiterpene alcohols identified as minor side products. Thus, the substitution of polar side chains for nonpolar side chains in terpenoid cyclase active sites can result in the stabilization of bound water molecules that, in turn, can serve template functions in isoprenoid cyclization reactions.

Structure of Sesquisabinene Synthase 1, a Terpenoid Cyclase That Generates a Strained [3.1.0] Bridged-Bicyclic Product.

The natural product sesquisabinene is a key component of the fragrant essential oil of the sandalwood tree, currently valued at $5,000/L. Sesquisabinene contains a highly strained [3.1.0] bicyclic ring system and is generated from farnesyl diphosphate in a reaction catalyzed by a class I terpenoid cyclase. To understand how the enzyme directs the formation of a strained hydrocarbon ring system, we now report the X-ray crystal structure of sesquisabinene synthase 1 (SQS1) from the Indian sandalwood tree ( Santalum album). Specifically, we report the structure of unliganded SQS1 at 1.90 Å resolution and the structure of its complex with three Mg2+ ions and the inhibitor ibandronate at 2.10 Å resolution. The bisphosphonate group of ibandronate coordinates to all three metal ions and makes hydrogen bond interactions with basic residues at the mouth of the active site. These interactions are similarly required for activation of the substrate diphosphate group to initiate catalysis, although partial occupancy binding of the Mg2+B ion suggests that this structure represents the penultimate metal coordination complex just prior to substrate activation. The structure of the liganded enzyme enables a precise definition of the enclosed active site contour that serves as a template for the cyclization reaction. This contour is very product-like in shape and readily fits an extended conformation of sesquisabinene and its precursor, the homobisabolyl cation. Structural comparisons of SQS1 with epi-isozizaene synthase mutants that also generate sesquisabinene suggest that [3.1.0] ring formation is not dependent on the isoprenoid tail conformation of the homobisabolyl cation.

＜Editors' Choice＞ Pulmonary Hypertension: Diagnosis, Management, and Treatment.

ABSTRACTPulmonary hypertension (PH) is a hemodynamic state that is characterized by a resting mean pulmonary artery pressure ≧ 25 mmHg. The common forms of PH are pulmonary arterial hypertension (PAH), chronic thromboembolic pulmonary hypertension (CTEPH), PH caused by left-heart disease, and PH due to lung disease. Previously regarded as untreatable, the treatment of PAH has dramatically advanced since the introduction of the drug epoprostenol in 1999, with three-year survival rates improving from 30%–40% to over 85%. Drugs available for the specific treatment of PAH include endothelin-receptor antagonists, phosphodiesterase type 5 inhibitors, soluble guanylate cyclase stimulators, prostacyclin analogs, and prostacyclin-receptor agonists. In the past decade, management and treatment of CTEPH have also improved. While pulmonary endarterectomy used to be the only option for the treatment of CTEPH, newer treatments include a soluble guanylate cyclase stimulator, which has proven to be an efficacious targeted therapy. Other cases benefit from balloon pulmonary angioplasty.

S-Nitrosylation of Clopidogrel inhibits LPS induced macrophage activation through NO/soluble Guanylate Cyclase pathways

S-Nitrosylation of Clopidogrel inhibits LPS induced macrophage activation through NO/soluble Guanylate Cyclase pathways

Synthesis of phenylpyrimidinones as guanylyl cyclase C inhibitors.

Diarrhea is one of the most important causes of mortality in the developing world, being responsible for 2.5 million deaths each year. Many of these deaths are caused by enterotoxigenic strains of bacteria, like Escherichia coli, that produce enterotoxins that cause acute watery diarrhea, commonly defined as secretory diarrhea. Studies on symptomatic patients indicate a high prevalence of enterotoxigenic E. coli strains producing the heat-stable toxin, STa. STa is a small, cysteine-rich peptide that binds to the extracellular receptor domain of guanylyl cyclase C (GCC), located at the luminal membrane of intestinal epithelial cells. GCC and its endogenous peptide ligands, guanylin and uroguanylin, play a key role in balancing water absorption and hydration of the intestinal lumen, as exemplified by the finding that loss of GCC function causes severe dehydration of the intestinal lumen, culminating in intestinal obstruction. From a mechanistic viewpoint, reduction of GCC activity offers an efficient approach to limit enterotoxigenic E. coli- provoked secretory diarrhea. Inhibition of GCC-mediated cGMP production would not only reduce anion secretion, but would also restore NHE3 activity, resulting in a comprehensive antidiarrheal action. In the present study, two novel phenylpyrimidinone derivatives were simultaneously synthesized and tested for their ability to block STa-induced CFTR activity in T84 cells.

CdaR-DAC signaling systems regulating the synthesis of a second messenger c-di-AMP in Leptospira interrogans

Objective To analyze the activity of diadenylate cyclase (DAC) encoded by LA3304 gene of Leptospira interrogans (L.interrogans) and to investigate the influence of CdaR encoded by LA3303 gene on DAC activity. Methods The LA3304 gene in L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was amplified by PCR and inserted into a prokaryotic expression system for expressing DAC. The expressed recombinant protein, rDAC, was purified by Ni-NTA affinity chromatography. High Performance Liquid Chromatography (HPLC) was used to measure the synthesis of c-di-AMP from ATP by rDAC in vitro. Bacterial two-hybrid analysis was used to detect the interaction between CdaR and DAC. Prokaryotic co-expression system was constructed and used in combination with HPLC to analyze the role of CdaR in activating DAC. Results The constructed prokaryotic expression system for LA3304 gene of L. interrogans strain Lai could highly express the rDAC upon the induction of IPTG. The purified rDAC showed high purity with a single protein band in gel as indicated by SDS-PAGE. rDAC could synthesize c-di-AMP from ATP in vitro. CdaR interacted with DAC and enhanced the activity of DAC (P<0.05). Conclusion DAC encoded by LA3304 gene was a diadenylate cyclase that could convert ATP into c-di-AMP. CdaR promoted the activation of DAC and formed a CdaR-DAC system with DAC. The system was involved in the synthesis of c-di-AMP in L. interrogans. Key words: Leptospira; c-di-AMP; DAC; CdaR

Eudesmane-type sesquiterpene diols directly synthesized by a sesquiterpene cyclase in Tripterygium wilfordii.

Cryptomeridiol, a typical eudesmane diol, is the active principle component of the antispasmodic Proximol. Although it has been used for many years, the biosynthesis pathway of cryptomeridiol has remained blur. Among terpenoid natural products, terpenoid cyclases are responsible for cyclization and generation of hydrocarbon backbones. The cyclization is mediated by carbocationic cascades and ultimately terminated via deprotonation or nucleophilic capture. Isoprene precursors are, respectively, converted into hydrocarbons or hydroxylated backbones. A sesquiterpene cyclase in Tripterygium wilfordii (TwCS) was determined to directly catalyze (E,E)-farnesyl pyrophosphate (FPP) to unexpected eudesmane diols, primarily cryptomeridiol. The function of TwCS was characterized by a modular pathway engineering system in Saccharomyces cerevisiae The major product determined by NMR spectroscopy turned out to be cryptomeridiol. This unprecedented production was further investigated in vitro, which verified that TwCS can directly produce eudesmane diols from FPP. Some key residues for TwCS catalysis were screened depending on the molecular model of TwCS and mutagenesis studies. As cryptomeridiol showed a small amount of volatile and medicinal properties, the biosynthesis of cryptomeridiol was reconstructed in S. cerevisiae Optimized assays including modular pathway engineering and the CRISPR-cas9 system were successfully used to improve the yield of cryptomeridiol in the S. cerevisiae The best engineered strain TE9 (BY4741 erg9::Δ-200-176 rox1::mut/pYX212-IDI + TwCS/p424-tHMG1) ultimately produced 19.73 mg/l cryptomeridiol in a shake flask culture.

Genome Mining and Comparative Biosynthesis of Meroterpenoids from Two Phylogenetically Distinct Fungi

AbstractTwo homologous meroterpenoid gene clusters consisting of contiguous genes encoding polyketide synthase (PKS), prenyltransferase (PT), terpenoid cyclase (TC) and other tailoring enzymes were identified from two phylogenetically distinct fungi through computational analysis. Media optimization guided by reverse‐transcription PCR (RT‐PCR) enabled two strains to produce eight new and two known meroterpenoids (1–10). Using gene inactivation, heterologous expression, and biochemical analyses, we revealed a new polyketide‐terpenoid assembly line that utilizes a pair of PKSs to synthesize 2,4‐dihydroxy‐6‐alkylbenzoic acid, followed by oxidative decarboxylation, farnesyl transfer, and terpene cyclization to construct the meroterpenoid scaffold. In addition, two of the isolated meroterpenoids (3 and 17 d) showed immunosuppressive bioactivity. Our work reveals a new strategy for meroterpenoid natural products discovery, and reveals the biosynthetic pathway for compounds 1–10.