Ternary complexes of isopentenyl phosphate kinase from Thermococcus paralvinellae reveal molecular determinants of non-natural substrate specificity.

Isopentenyl phosphate kinases (IPKs) have recently garnered attention for their central role in biocatalytic "isoprenol pathways," which seek to reduce the synthesis of the isoprenoid precursors to two enzymatic steps. Furthermore, the natural promiscuity of IPKs toward non-natural alkyl-monophosphates (alkyl-Ps) as substrates has hinted at the isoprenol pathways' potential to access novel isoprenoids with potentially useful activities. However, only a handful of IPK crystal structures have been solved to date, and even fewer of these contain non-natural substrates bound in the active site. The current study sought to elucidate additional ternary complexes bound to non-natural substrates using the IPK homolog from Thermococcus paralvinellae (TcpIPK). Four such structures were solved, each bound to a different non-natural alkyl-P and the phosphoryl donor substrate/product adenosine triphosphate (ATP)/adenosine diphosphate (ADP). As expected, the quaternary, tertiary, and secondary structures of TcpIPK closely resembled those of IPKs published previously, and kinetic analysis of a novel alkyl-P substrate highlighted the potentially dramatic effects of altering the core scaffold of the natural substrate. Even more interesting, though, was the discovery of a trend correlating the position of two α helices in the active site with the magnitude of an IPK homolog's reaction rate for the natural reaction. Overall, the current structures of TcpIPK highlight the importance of continued structural analysis of the IPKs to better understand and optimize their activity with both natural and non-natural substrates.

Comparison of Chemical Composition between Kuromoji (Lindera umbellata) Essential Oil and Hydrosol and Determination of the Deodorizing Effect.

Kuromoji (Lindera umbellata) is a tree that grows throughout Japan. The components of kuromoji essential oil have antitumor and aromatherapy effects. However, the composition of the hydrosol, obtained as a by-product of the essential oil process, is unknown. Furthermore, it is unknown whether kuromoji essential oil has a deodorizing effect. Therefore, the purpose of the current study was to compare the chemical composition of kuromoji essential oil and hydrosol, as well as evaluate the deodorizing effect of the former. The chemical composition of samples was evaluated using gas chromatography-mass spectrometry (GC-MS). Additionally, the deodorizing effect of Kuromoji essential oil was investigated with the detector tube method using ammonia, hydrogen sulfide, methyl mercaptan, and isovaleric acid. Linalool was the most abundant component in both the essential oil and hydrosol; however, its proportion was higher in the hydrosol (57.5%) than in the essential oil (42.8%). The hydrosol contained fewer chemical components, but higher proportions of trans-geraniol and ethanol. Moreover, the essential oil eliminated 50% of ammonia and 97.6% or more of isovaleric acid. Interestingly, linalool was soluble in the hydrosol and did not irritate the skin. This suggests that the hydrosol may be an effective foot care product.

The Beneficial Effects of Morusin, an Isoprene Flavonoid Isolated from the Root Bark of Morus.

The root bark of Morus has long been appreciated as an antiphlogistic, diuretic and expectorant drug in Chinese herbal medicine, albeit with barely known targets and mechanisms of action. In the 1970s, the development of analytic chemistry allowed for the discovery of morusin as one of 7 different isoprene flavonoid derivatives in the root bark of Morus. However, the remarkable antioxidant capacity of morusin with the unexpected potential for health benefits over the other flavonoid derivatives has recently sparked scientific interest in the biochemical identification of target proteins and signaling pathways and further clinical relevance. In this review, we discuss recent advances in the understanding of the functional roles of morusin in multiple biological processes such as inflammation, apoptosis, metabolism and autophagy. We also highlight recent in vivo and in vitro evidence on the clinical potential of morusin treatment for multiple human pathologies including inflammatory diseases, neurological disorders, diabetes, cancer and the underlying mechanisms.

Molecular Mechanisms of Natural Rubber Biosynthesis.

Natural rubber (NR), principally comprising cis-1,4-polyisoprene, is an industrially important natural hydrocarbon polymer because of its unique physical properties, which render it suitable for manufacturing items such as tires. Presently, industrial NR production depends solely on latex obtained from the Pará rubber tree, Hevea brasiliensis. In latex, NR is enclosed in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The similarity of the basic carbon skeleton structure between NR and dolichols and polyprenols, which are found in most organisms, suggests that the NR biosynthetic pathway is related to the polyisoprenoid biosynthetic pathway and that rubber transferase, which is the key enzyme in NR biosynthesis, belongs to the cis-prenyltransferase family. Here, we review recent progress in the elucidation of molecular mechanisms underlying NR biosynthesis through the identification of the enzymes that are responsible for the formation of the NR backbone structure.

Ammonia uptake by transmembrane pH gradient poly(isoprene)-block-poly(ethylene glycol) polymersomes.

Transmembrane pH gradient poly(isoprene)-block-poly(ethylene glycol) (PI-b-PEG) polymersomes were investigated for their potential use in the detoxification of ammonia, a metabolite that is excessively present in patients suffering from urea cycle disorders and advanced liver diseases, and which causes neurotoxic effects (e.g., hepatic encephalopathy). Polymers varying in PI and PEG block length were synthesized via nitroxide-mediated polymerization and screened for their ability to self-assemble into polymersomes in aqueous media. Ammonia sequestration by the polymersomes was investigated in vitro. While most vesicular systems were able to capture ammonia in simulated intestinal fluids, uptake was lost in partially dehydrated medium mimicking conditions in the colon. Polymeric crosslinking of residual olefinic bonds in the PI block increased polymersome stability, partially preserving the ammonia capture capacity in the simulated colon environment. These more stable vesicular systems hold promise for the chronic oral treatment of hyperammonemia.

Isoprene-Derived Secondary Organic Aerosol Induces the Expression of MicroRNAs Associated with Inflammatory/Oxidative Stress Response in Lung Cells.

Exposure to fine particulate matter (PM2.5), of which secondary organic aerosol (SOA) is a major constituent, is linked to adverse health outcomes, including cardiovascular disease, lung cancer, and preterm birth. Atmospheric oxidation of isoprene, the most abundant nonmethane hydrocarbon emitted into Earth's atmosphere primarily from vegetation, contributes to SOA formation. Isoprene-derived SOA has previously been found to alter inflammatory/oxidative stress genes. MicroRNAs (miRNAs) are epigenetic regulators that serve as post-transcriptional modifiers and key mediators of gene expression. To assess whether isoprene-derived SOA alters miRNA expression, BEAS-2B lung cells were exposed to laboratory-generated isoprene-derived SOA constituents derived from the acid-driven multiphase chemistry of authentic methacrylic acid epoxide (MAE) or isomeric isoprene epoxydiols (IEPOX) with acidic sulfate aerosol particles. These IEPOX- and MAE-derived SOA constituents have been shown to be measured in large quantities within PM2.5 collected from isoprene-rich areas affected by acidic sulfate aerosol particles derived from human activities. A total of 29 miRNAs were identified as differentially expressed when exposed to IEPOX-derived SOA and 2 when exposed to MAE-derived SOA, a number of which are inflammatory/oxidative stress associated. These results suggest that miRNAs may modulate the inflammatory/oxidative stress response to SOA exposure, thereby advancing the understanding of airway cell epigenetic response to SOA.

Highly Active Iminopyridyl Iron-Based Catalysts for the Polymerization of Isoprene.

A series of iminopyridyl-based ligands, 6-[(Ar)N=C(R)]-2-C6H5N [(Ar = 2,6-Me2-C6H3, R = Me (L1); Ar = 2,6-iPr2-C6H3, R = Me (L2); Ar = 2,6-Me2-C6H3, R = H (L3); Ar = 2,6-iPr2-C6H3, R = H (L4); Ar = 3,5-(CF3)2-C6H3, R = Me (L5); Ar = C6F5, R = Me (L6)], and their corresponding iron (II) complexes were developed to investigate their application in the controlled coordinative polymerization of isoprene. The modulation of steric and electronic properties within this family of ligands/pre-catalysts has shown to influence the stereo-selectivity and activity of the polymerization of isoprene after activation. Upon activation with various co-catalysts such as AliBu3/[Ph3C][B(C6F5)4], AlEt3/[Ph3C][B(C6F5)4] or MAO, the resulting catalysts produced polyisoprenes with an excellent conversion (>99% of 500-5000 equiv.) within less than 1 h (TOF > 500 h-1) and having a variety of stereo-/regio-regularities. The presence of electron-donating and withdrawing groups drastically impacted the activity and the stereoselectivity of the catalysts during the course of the polymerization of isoprene. When activated with AliBu3/[Ph3C][B(C6F5)4], the complexes {6-[(2,6-Me2-C6H3)N=C(Me)]-2-C6H5N}FeCl2 (C1) and {6-[(2,6-iPr2-C6H3)N=C(Me)]-2-C6H5N}FeCl2 (C2) exhibited moderate trans-1,4 selectivity (>67%) while the iron-based systems bearing related aldiminopyridyl ligands {6-[(2,6-Me2-C6H3)N=C(H)]-2-C6H5N}FeCl2 (C3) and {6-[(2,6-iPr2-C6H3)N=C(H)]-2-C6H5N}FeCl2 (C4) were found to afford significant cis-1,4 selectivity at low temperature (>86% at -40 °C). On the other hand, the ternary {6-[(3,5-(CF3)2-C6H3)N=C(Me)]-2-C6H5N}FeCl2 (C5) or {6-[(C6F5)N=C(Me)]-2-C6H5N}FeCl2 (C6)/AliBu3/[Ph3C][B(C6F5)4] catalytic combinations showed exceptional activity for the polymerization of isoprene (TOF > 1,000,000 h-1), albeit providing less stereoselectivity.

Terpenes, hormones and life: isoprene rule revisited.

The year 2019 marks the 80th anniversary of the 1939 Nobel Prize in Chemistry awarded to Leopold Ruzicka (1887-1976) for work on higher terpene molecular structures, including the first chemical synthesis of male sex hormones. Arguably his crowning achievement was the 'biogenetic isoprene rule', which helped to unravel the complexities of terpenoid biosynthesis. The rule declares terpenoids to be enzymatically cyclized products of substrate alkene chains containing a characteristic number of linear, head-to-tail condensed, C5 isoprene units. The number of repeat isoprene units dictates the type of terpene produced (i.e., 2, monoterpene; 3, sesquiterpene; 4, diterpene, etc.). In the case of triterpenes, six C5 isoprene units combine into C30 squalene, which is cyclized into one of the signature carbon skeletons from which myriad downstream triterpenoid structures are derived, including sterols and steroids. Ruzicka also had a keen interest in the origin of life, but the pivotal role of terpenoids has generally been overshadowed by nucleobases, amino acids, and sugars. To redress the balance, we provide a historical and evolutionary perspective. We address the potential abiotic generation of isoprene, the crucial role that polyprene terpenoids played in early membranes and cellular life, and emphasize that endocrinology from microbes to plants and vertebrates is firmly grounded on Ruzicka's pivotal insights into the structure and function of terpenes. A harmonizing feature is that all known lifeforms (including bacteria) biosynthesize triterpenoid substances that are essential for cellular membrane formation and function, from which signaling molecules such as steroid hormones and cognate receptors are likely to have evolved.

Development of novel on-line capillary gas chromatography-based analysis method for volatile organic compounds produced by aerobic fermentation.

Many volatile compounds, such as isoprene, a precursor used in the synthesis of natural rubber, have been produced through fermentation using genetically engineered microorganisms. Despite this biotechnological success, measuring the concentrations of volatile compounds during fermentation is difficult because of their high volatility. In current systems, off-line analytical methods usually lead to product loss, whereas on-line methods raise the production cost due to the requirement of complex devices. Here, we developed a novel on-line gas chromatography (GC)-based system for analyzing the concentration of isoprene with the aim to minimize the cost and requirement for devices as compared to current strategies. In this system, a programmable logic controller is used to combine conventional GC with a syringe pump module (SPM) directly connected to the exhaust pipe of the fermentor, and isoprene-containing samples are continuously pumped from the SPM into the GC using an air cylinder recycle stream. We showed that this novel system enables isoprene analysis during fermentation with convenient equipment and without the requirement of an expensive desorption tube. Furthermore, this system may be extended to the detection of other volatile organic compounds in fermentation or chemical processes.

A Single Amino Acid Switch Alters the Isoprene Donor Specificity in Ribosomally Synthesized and Post-Translationally Modified Peptide Prenyltransferases.

Mutation at a single amino acid alters the isoprene donor specificity of prenyltransferases involved in the modification of ribosomally synthesized and post-translationally modified peptides (RiPPs). Though most characterized RiPP prenyltransferases carry out the regiospecific transfer of C5 dimethylallyl donor to the side chain atoms on macrocyclic acceptor substrates, the elucidation of the cyanobactin natural product piricyclamide 70005E1 identifies an O-geranyl modification on Tyr, a reaction with little prior biochemical precedence. Reconstitution and kinetic studies of the presumptive geranyltransferase PirF shows that the enzyme utilizes a C10 donor, with no C5 transferase activity. The crystal structure of PirF reveals a single amino acid difference in the vicinity of the isoprene-binding pocket, relative to the C5 utilizing enzymes. Remarkably, only a single amino acid mutation is necessary to completely switch the donor specificity from a C5 to a C10 prenyltransferase, and vice versa. Lastly, we demonstrate that these enzymes may be used for the chemospecific attachment of C5 or C10 lipid groups on lanthipeptides, an unrelated class of RiPP natural products. These studies represent a rare example where prenyl donor specificity can be discretely altered, which expands the arsenal of synthetic biology tools for tuning biological activities of peptide natural products.

Hypermonins A and B, two 6-norpolyprenylated acylphloroglucinols with unprecedented skeletons from Hypericum monogynum.

Two new 6-norpolycyclic polyprenylated acylphloroglucinols (PPAPs), hypermonins A (1) and B (2), featuring an undescribed decahydroindeno[1,7-bc]furan ring system, were isolated from the leaves and twigs of Hypericum monogynum. These compounds are a pair of epimers with opposite configurations at the C-5 position. Their structures, including their absolute configurations, were determined by extensive spectroscopic analysis and electronic circular dichroism (ECD) calculations. A plausible biosynthetic pathway of 1 and 2 was also proposed. Compound 1 exhibited a significant protective effect against corticosterone-induced injury in PC12 cells.

Effect of secondary organic aerosol from isoprene-derived hydroxyhydroperoxides on the expression of oxidative stress response genes in human bronchial epithelial cells.

Isoprene-derived secondary organic aerosol (SOA), which comprise a large portion of atmospheric fine particulate matter (PM2.5), can be formed through various gaseous precursors, including isoprene epoxydiols (IEPOX), methacrylic acid epoxide (MAE), and isoprene hydroxyhydroperoxides (ISOPOOH). The composition of the isoprene-derived SOA affects its reactive oxygen species (ROS) generation potential and its ability to alter oxidative stress-related gene expression. In this study we assess effects of isoprene SOA derived solely from ISOPOOH oxidation on human bronchial epithelial cells by measuring the gene expression changes in 84 oxidative stress-related genes. In addition, the thiol reactivity of ISOPOOH-derived SOA was measured through the dithiothreitol (DTT) assay. Our findings show that ISOPOOH-derived SOA alter more oxidative-stress related genes than IEPOX-derived SOA but not as many as MAE-derived SOA on a mass basis exposure. More importantly, we found that the different types of SOA derived from the various gaseous precursors (MAE, IEPOX, and ISOPOOH) have unique contributions to changes in oxidative stress-related genes that do not total all gene expression changes seen in exposures to atmospherically relevant compositions of total isoprene-derived SOA mixtures. This study suggests that amongst the different types of known isoprene-derived SOA, MAE-derived SOA are the most potent inducer of oxidative stress-related gene changes but highlights the importance of considering isoprene-derived SOA as a total mixture for pollution controls and exposure studies.

Isotactic and Syndiotactic Alternating Ethylene/Propylene Copolymers Obtained Through Non-Catalytic Hydrogenation of Highly Stereoregular cis-1,4 Poly(1,3-diene)s.

The homogeneous non-catalytic hydrogenation of cis-1,4 poly(isoprene), isotactic cis-1,4 poly(1,3-pentadiene) and syndiotactic cis-1,4 poly(1,3-pentadiene) with diimide, formed by thermal decomposition of para-toluenesulfonylhydrazide, is examined. Perfectly alternating ethylene/propylene copolymers having different tacticity (i.e., isotactic and syndiotactic), which are difficult to synthesize by stereospecific copolymerization of the corresponding monomers, are obtained. Both isotactic and syndiotactic alternating ethylene/propylene copolymers are amorphous, with very low glass transition temperatures.

Hepatoprotective hemiterpene glycosides from the rhizome of Cibotium barometz (L.) J. Sm.

Five previously undescribed hemiterpene glycosides, cibotiumbarosides E-I, and two known hemiterpene glucosides, were isolated from the rhizome of Cibotium barometz (L.) J. Sm. The structures of cibotiumbarosides E-I were established by 1D and 2D NMR spectroscopic analyses and HRMS. The absolute configuration of the aglycone of cibotiumbaroside E was assigned by calculated ECD with the TDDFT method. Cibotiumbarosides F and I both exhibited remarkable hepatoprotective activity against APAP-induced acute liver damage in vitro, which were more effective than the positive control, bicyclol. On the other hand, seven hemiterpene glycosides were all inactive in assays of cytotoxicity, neuroprotection, antidiabetes and anti-inflammation.

Polyisoprenylated benzoylphloroglucinol derivatives from Hypericum scabrum.

Four new polyisoprenylated benzoylphloroglucinol derivatives, hyperscabrones J-M (1-4), were isolated from the air-dried aerial parts of Hypericum scabrum. Their structures were elucidated by spectroscopic methods and were subsequently confirmed by comparing with data of known compounds. The absolute configuration of the bicyclo[3.3.1]nonane-2,4,9-trione core was defined by the experimental and calculated electronic circular dichroism (ECD) spectra. The evaluation of their hepatoprotective activities against paracetamol-induced HepG2 cell damage showed that compounds 2 and 4 exhibited significant hepatoprotection at 10µM.

The antiangiogenic effects of polyisoprenylated cysteinyl amide inhibitors in HUVEC, chick embryo and zebrafish is dependent on the polyisoprenyl moiety.

Angiogenesis is essential for solid tumor growth, therapeutic resistance and metastasis, the latest accounting for 90% of cancer deaths. Although angiogenesis is essential for the malignant transformations in solid tumors and therefore is an attractive target, few drugs are available that block tumor angiogenesis. The focus has been to block signaling by receptor tyrosine kinases (RTKs), such as for vascular endothelial growth factor (VEGF), whose activation abrogate apoptosis and promote angiogenesis. The polyisoprenylated cysteinyl amide inhibitors (PCAIs) were designed to modulate aberrant polyisoprenylated small G-proteins such as mutant Ras whose constitutive activation promotes RTKs signaling. Since polyisoprenylation is essential for protein-protein interactions and functions of G-proteins, we hypothesized that the PCAIs would disrupt the monomeric G-protein signaling thereby effectively inhibiting angiogenesis. In this study we determined the effects of PCAIs on human umbilical vein endothelial cells (HUVEC) tube formation, cell viability, cell migration and invasion as well as in vivo using the chick chorioallantoic membrane (CAM) and zebrafish models. At sub- to low micromolar concentrations, the PCAIs inhibit the native and VEGF-stimulated cell migration and invasion as well as tube formation and angiogenesis in CAM and zebrafish embryos. The concentrations that block the angiogenic processes were lower than those that induce cell death. Since angiogenesis is essential for tumor growth but otherwise limited to wound healing, feeding fat cells and uterine wall repair in adults, it is conceivable that these compounds can be developed into safer therapeutics for cancers and retinal neovascularization that leads to loss of vision.

Catalytic Enantioselective Conjugate Additions of (pin)B-Substituted Allylcopper Compounds Generated in situ from Butadiene or Isoprene.

Multicomponent catalytic enantioselective transformations that entail the combination of butadiene or isoprene (common feedstock), an enoate (prepared in one step) and B2 (pin)2 (commercially available) are presented. These processes constitute an uncommon instance of conjugate addition of an allyl moiety and afford the desired products in up to 83 % yield and 98:2 enantiomeric ratio. Based on DFT calculations stereochemical models and rationale for the observed profiles in selectivity are provided.

Two unexpected promiscuous activities of the iron-sulfur protein IspH in production of isoprene and isoamylene.

BACKGROUND: Bacillus species, possessing the methylerythritol phosphate (MEP) pathway for the synthesis of isoprenoid feedstock, are the highest producers of isoprene among bacteria; however, the enzyme responsible for isoprene synthesis has not been identified. The iron-sulfur protein IspH is the final enzyme of the MEP pathway and catalyses the reductive dehydration of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP) to form isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). In this study, we demonstrated two unexpected promiscuous activities of IspH from alkaliphilic Bacillus sp. N16-5, which can produce high levels of isoprene. RESULTS: Bacillus sp. N16-5 IspH could catalyse the formation of isoprene from HMBPP and the conversion of DMAPP into a mixture of 2-methyl-2-butene and 3-methyl-1-butene. Both reactions require an electron transfer system, such as that used for HMBPP dehydration. Isoprene and isoamylene synthesis in Bacillus sp. N16-5 was investigated and the reaction system was reconstituted in vitro, including IspH, ferredoxin and ferredoxin-NADP(+)-reductase proteins and NADPH. The roles of specific IspH protein residues were also investigated by site-directed mutagenesis experiments; two variants (H131N and E133Q) were found to have lost the HMBPP reductase activity but could still catalyse the formation of isoprene. Overexpression of IspH H131N in Bacillus sp. N16-5 resulted in a twofold enhancement of isoprene production, and the yield of isoprene from the strain expressing E133Q was increased 300% compared with the wild-type strain. CONCLUSIONS: IspH from Bacillus sp. N16-5 is a promiscuous enzyme that can catalyse formation of isoprene and isoamylene. This enzyme, especially the H131N and E133Q variants, could be used for the production of isoprene from HMBPP.

The potential of the mevalonate pathway for enhanced isoprenoid production.

The cytosol-localised mevalonic acid (MVA) pathway delivers the basic isoprene unit isopentenyl diphosphate (IPP). In higher plants, this central metabolic intermediate is also synthesised by the plastid-localised methylerythritol phosphate (MEP) pathway. Both MVA and MEP pathways conspire through exchange of intermediates and regulatory interactions. Products downstream of IPP such as phytosterols, carotenoids, vitamin E, artemisinin, tanshinone and paclitaxel demonstrate antioxidant, cholesterol-reducing, anti-ageing, anticancer, antimalarial, anti-inflammatory and antibacterial activities. Other isoprenoid precursors including isoprene, isoprenol, geraniol, farnesene and farnesol are economically valuable. An update on the MVA pathway and its interaction with the MEP pathway is presented, including the improvement in the production of phytosterols and other isoprenoid derivatives. Such attempts are for instance based on the bioengineering of microbes such as Escherichia coli and Saccharomyces cerevisiae, as well as plants. The function of relevant genes in the MVA pathway that can be utilised in metabolic engineering is reviewed and future perspectives are presented.

Phenolic constituents of the Bangladeshi medicinal plant Pothos scandens and their anti-estrogenic, hyaluronidase inhibition, and histamine release inhibitory activities.

Extracts from the stem and roots of the Bangladeshi medicinal plant Pothos scandens L. (Araceae) were isolated, and three hemiterpene glucoside aromatic esters, pothobanosides A (1), B (2), and C (3), and a phenylisobutanoid, pothobanol (4), along with 14 known compounds, were characterized. The isolates were tested for their estrogenic/anti-estrogenic activity using the estrogen-responsive human breast cancer cell lines MCF-7 and T47D, and syringoyl derivatives (2, 3, and canthoside B) showed strong inhibitory activity against both cell lines. Their less oxygenated analogs (1, and markhamioside F) were almost inactive. The isolates were also evaluated for hyaluronidase and histamine release inhibitory activities, and pothobanoside A (1) showed significant hyaluronidase inhibitory activity among the isolated compounds, which was similar to that of the positive control rosmarinic acid. Because hyaluronidase produces an angiogenic response that has been implicated in tumor invasiveness and metastasis, 1 could be valuable as an anti-tumor compound with a different mechanism of action from related compounds (2, 3). Pothobanoside C (3) and pothobanol (4) were also found to inhibit histamine release to a similar degree to the positive control epigallocatechin 3-O-(3"-O-methyl)-gallate. The histamine release inhibitory potency of these isolates may support the traditional uses of this plant in folk medicine.