Global transcriptome analyses and regulatory mechanisms in Halothece sp. PCC 7418 exposed to abiotic stresses.

Halotolerant species are of interest since they occur naturally in environments with excess toxic ions. The cyanobacterium Halothece sp. PCC 7418 (hereafter referred to as Halothece) exhibits remarkable halotolerance and was used to examine stress-responsive regulatory mechanisms. The effects of five different stimuli on Halothece transcriptomes were examined using RNA sequencing. In response to diverse stresses, there were both common and stress-specific transcriptional responses. A common upregulated gene set under all stresses consisted of nine differentially expressed genes (DEGs). We also found that osmotic stress elicited the largest set of DEGs. Salt- and osmotic-responsive regulatory mechanisms shared common pathways. DEGs that were upregulated under salt stress encoded proteins involved in photosynthesis and related machineries. Furthermore, DEGs encoding two-component system (TCS) factors, transcriptional factors, scaffolds for protein-protein interactions, transporters, protein turnover factors, and lipid biosynthesis enzymes were also identified under salt stress. Notably, one-carbon (1C) metabolism factors, glycine betaine (GB) synthesis enzymes, and GB transporters were upregulated under salt stress. Metabolic analyses revealed that GB accumulated under salt stress, while mycosporine-2-glycine (M2G) accumulated under salt or osmotic stress. None of the nutrient starvations induced GB nor M2G accumulation. These results suggested that GB and M2G are two osmoprotectants that contribute to halotolerance. Based on our results, we proposed regulatory mechanisms that are crucial for halotolerance, which are coordinated with the GB, M2G, 1C, amino acid, and central carbon interlinking metabolic pathways. 1C metabolism directly fulfills the high metabolite requirements for halotolerance together with the ancillary role of several metabolic pathways.Key Points• Global transcriptome surveys together with molecular and metabolite analyses provide insights into regulatory networks that are crucial for halotolerance• Regulatory networks that are crucial for halotolerance coordinated with the two key osmoprotectants, one carbon, amino acid, and central carbon interlinking metabolic pathways• The findings have translational relevance in genomic and transcriptomic mechanisms of halotolerance.

Experimental characterization of the association of ß-cyclodextrin and eight novel cyclodextrin derivatives with two guest compounds.

We investigate the binding of native ß-cyclodextrin (ß-CD) and eight novel ß-CD derivatives with two different guest compounds, using isothermal calorimetry and 2D NOESY NMR. In all cases, the stoichiometry is 1:1 and binding is exothermic. Overall, modifications at the 3' position of ß-CD, which is at the secondary face, weaken binding by several kJ/mol relative to native ß-CD, while modifications at the 6' position (primary face) maintain or somewhat reduce the binding affinity. The variations in binding enthalpy are larger than the variations in binding free energy, so entropy-enthalpy compensation is observed. Characterization of the bound conformations with NOESY NMR shows that the polar groups of the guests may be situated at either face, depending on the host molecule, and, in some cases, both orientations are populated. The present results were used in the SAMPL7 blinded prediction challenge whose results are detailed in the same special issue of JCAMD.

Cysteine Nucleophiles in Glycosidase Catalysis: Application of a Covalent ß-l-Arabinofuranosidase Inhibitor.

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3 (Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived ß-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This ß-l-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.

Synthetic Cannabinoid Hydroxypentyl Metabolites Retain Efficacy at Human Cannabinoid Receptors.

Synthetic cannabinoids (SCs) are novel psychoactive substances that are easily acquired, widely abused as a substitute for cannabis, and associated with cardiotoxicity and seizures. Although the structural bases of these compounds are scaffolds with known affinity and efficacy at the human cannabinoid type-1 receptor (hCB1), upon ingestion or inhalation they can be metabolized to multiple chemical entities of unknown pharmacological activity. A large proportion of these metabolites are hydroxylated on the pentyl chain, a key substituent that determines receptor affinity and selectivity. Thus, the pharmacology of SC metabolites may be an important component in understanding the in vivo effects of SCs. We examined nine SCs (AB-PINACA, 5F-AB-PINACA, ADB/MDMB-PINACA, 5F-ADB, 5F-CUMYL-PINACA, AMB-PINACA, 5F-AMB, APINACA, and 5F-APINACA) and their hydroxypentyl (either 4-OH or 5-OH) metabolites in [3H]CP55,940 receptor binding and the [35S]GTPγS functional assay to determine the extent to which these metabolites retain activity at cannabinoid receptors. All of the SCs tested exhibited high affinity (<10 nM) and efficacy for hCB1 and hCB2 The majority of the hydroxypentyl metabolites retained full efficacy at hCB1 and hCB2, albeit with reduced affinity and potency, and exhibited greater binding selectivity for hCB2 These data suggest that phase I metabolites may be contributing to the in vivo pharmacology and toxicology of abused SCs. Considering this and previous reports demonstrating that metabolites retain efficacy at the hCB1 receptor, the full pharmacokinetic profiles of the parent compounds and their metabolites need to be considered in terms of the pharmacological effects and time course associated with these drugs.

Cystobasidium keelungensis sp. nov., a novel mycosporine producing carotenogenic yeast isolated from the sea surface microlayer in Taiwan.

Cystobasidium keelungensis SN2T (CBS 6949 = BCRC 920080), a new anamorphic basidiomycetous yeast species, is described in this paper. The strains belonging to this species were isolated from the sea surface microlayer and underlying water in Taiwan. These strains were identified by examining nucleotide sequences in the species-specific D1/D2 domains of the large subunit (LSU) ribosomal RNA (rRNA) and by evaluating their physiological characteristics. Phylogenetic analyses of D1/D2 sequences revealed that C. keelungensis was most closely related to the species C. slooffiae (LSU rRNA gene sequence divergence of 1.5%), and it belonged to the Erythrobasidium clade. No sexual reproduction was observed. This species differed from related species in carbon and nitrogen assimilation patterns and growth at 35 °C. Screening C. keelungensis for the presence of UV-absorbing compounds showed that mycosporine-glutaminol-glucoside and mycosporine-glutamicol-glucoside (maximum absorption: 310 nm) were the major UV-absorbing compounds, which differ from the compounds present in some freshwater yeast strains reported in previous studies. After UV induction, SN2 had a higher level of mycosporine production than other carotenogenic yeasts in this study.

Two divergent Symbiodinium genomes reveal conservation of a gene cluster for sunscreen biosynthesis and recently lost genes.

BACKGROUND: The marine dinoflagellate, Symbiodinium, is a well-known photosynthetic partner for coral and other diverse, non-photosynthetic hosts in subtropical and tropical shallows, where it comprises an essential component of marine ecosystems. Using molecular phylogenetics, the genus Symbiodinium has been classified into nine major clades, A-I, and one of the reported differences among phenotypes is their capacity to synthesize mycosporine-like amino acids (MAAs), which absorb UV radiation. However, the genetic basis for this difference in synthetic capacity is unknown. To understand genetics underlying Symbiodinium diversity, we report two draft genomes, one from clade A, presumed to have been the earliest branching clade, and the other from clade C, in the terminal branch. RESULTS: The nuclear genome of Symbiodinium clade A (SymA) has more gene families than that of clade C, with larger numbers of organelle-related genes, including mitochondrial transcription terminal factor (mTERF) and Rubisco. While clade C (SymC) has fewer gene families, it displays specific expansions of repeat domain-containing genes, such as leucine-rich repeats (LRRs) and retrovirus-related dUTPases. Interestingly, the SymA genome encodes a gene cluster for MAA biosynthesis, potentially transferred from an endosymbiotic red alga (probably of bacterial origin), while SymC has completely lost these genes. CONCLUSIONS: Our analysis demonstrates that SymC appears to have evolved by losing gene families, such as the MAA biosynthesis gene cluster. In contrast to the conservation of genes related to photosynthetic ability, the terminal clade has suffered more gene family losses than other clades, suggesting a possible adaptation to symbiosis. Overall, this study implies that Symbiodinium ecology drives acquisition and loss of gene families.

CYP76C1 (Cytochrome P450)-Mediated Linalool Metabolism and the Formation of Volatile and Soluble Linalool Oxides in Arabidopsis Flowers: A Strategy for Defense against Floral Antagonists.

The acyclic monoterpene alcohol linalool is one of the most frequently encountered volatile compounds in floral scents. Various linalool oxides are usually emitted along with linalool, some of which are cyclic, such as the furanoid lilac compounds. Recent work has revealed the coexistence of two flower-expressed linalool synthases that produce the (S)- or (R)-linalool enantiomers and the involvement of two P450 enzymes in the linalool oxidation in the flowers of Arabidopsis thaliana. Partially redundant enzymes may also contribute to floral linalool metabolism. Here, we provide evidence that CYP76C1 is a multifunctional enzyme that catalyzes a cascade of oxidation reactions and is the major linalool metabolizing oxygenase in Arabidopsis flowers. Based on the activity of the recombinant enzyme and mutant analyses, we demonstrate its prominent role in the formation of most of the linalool oxides identified in vivo, both as volatiles and soluble conjugated compounds, including 8-hydroxy, 8-oxo, and 8-COOH-linalool, as well as lilac aldehydes and alcohols. Analysis of insect behavior on CYP76C1 mutants and in response to linalool and its oxygenated derivatives demonstrates that CYP76C1-dependent modulation of linalool emission and production of linalool oxides contribute to reduced floral attraction and favor protection against visitors and pests.

Effects of morphine/CP55940 mixtures on an impulsive choice task in rhesus monkeys.

µ-Opioid receptor agonists are commonly used to treat pain despite their adverse effects. In preclinical studies, cannabinoid receptor agonists increase the potency of opioids for producing antinociceptive but not reinforcing effects. It is unknown whether other adverse effects of these drugs, such as impairment of complex behavior, are enhanced by their co-administration. This study characterized the effects of morphine (µ-opioid receptor agonist; 0.32-5.6 mg/kg, subcutaneously) and CP55940 (CB1/CB2 cannabinoid receptor agonist; 0.0032-0.32 mg/kg, subcutaneously), alone and in mixtures, in monkeys (n=3) choosing between one pellet delivered immediately and two pellets delivered after a delay. Two consecutive choices of the immediate or delayed reward decreased or increased, respectively, the delay. The median adjusted delay, indicating indifference between the immediate and delayed reinforcers, was increased by morphine (3.2 mg/kg) and CP55940 (0.01-0.032 mg/kg). Performance after administration of morphine (0.32 and 1 mg/kg)/CP55940 (0.0032-0.032 mg/kg) mixtures was not different from performance after CP55940 alone. Neither morphine, CP55940, nor mixtures decreased the median adjusted delay (i.e. increased impulsivity). These findings failed to confirm previous studies showing that morphine increases impulsivity, perhaps because of procedural differences among studies. Treatment of pain often requires repeated drug administration; thus, it remains to be determined whether the present findings predict the effects of chronically administered morphine/CP5540 mixtures on impulsive choice.

Inhibitory effects of mycosporine-2-glycine isolated from a halotolerant cyanobacterium on protein glycation and collagenase activity.

Mycosporine-2-glycine (M2G), isolated from the halotolerant cyanobacterium Aphanothece halophytica, was purified and characterized in order to determine its utility as a cosmetic and pharmaceutical ingredient. M2G efficiently inhibited protein crosslinking. The inhibitory activity of M2G was significantly greater than that of the well-known Maillard reaction inhibitor aminoguanidine. In addition, M2G and other known mycosporine-like amino acids inhibited bacterial collagenase activity. To the best of our knowledge, this is the first report describing that M2G specifically inhibits the formation of advanced glycation end-products (AGEs), which play a critical role in ageing process and age-related diseases. These observations indicate that M2G may have potential therapeutic applications by suppressing the formation of AGEs and inhibiting excess collagenase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycosporine-like amino acids (MAAs) are known as multifunctional natural compounds. The MAA mycosporine-2-glycine (M2G), isolated from the halotolerant cyanobacterium Aphanothece halophytica, has potential therapeutic applications for the prevention of skin ageing. Purified M2G was endotoxin-free. M2G had greater inhibitory activity of protein cross-linking compared with well-known inhibitor, aminoguanidine and hindered bacterial collagenase activity. The mechanisms for these inhibitory activities of M2G are discussed in this study.

Volatile organic compounds from Hypoxylon anthochroum endophytic strains as postharvest mycofumigation alternative for cherry tomatoes.

The antifungal activity and chemical composition of the volatile organic compounds (VOCs) produced by four Hypoxylon anthochroum endophytic strains were analyzed. The bioactivity of the VOCs synthesized at different periods of incubation on rice medium was assessed, both in vivo and in vitro, against the phytopathogen Fusarium oxysporum. The in vivo effect was evaluated on cherry tomatoes, while the mechanism of action was determined in vitro analyzing the phytopathogen's growth, respiration and cell membrane permeability. In general, the VOCs from all strains and incubation periods significantly inhibited the growth of F. oxysporum on cherry tomatoes with percentages over 50%. They significantly inhibited the pathogen growth and respiration, and altered the cell membrane permeability and hyphal morphology. The chemical composition was analyzed after solid phase microextraction. In total, 36 VOCs were identified in the four strains, mainly sesquiterpenes and monoterpenes. Among the monoterpenes, eucalyptol had the highest fiber affinity (>60% area) in three of the four strains studied; thus, it could be considered as a chemical marker for H. antochroum. Chemical markers are important for the identification and differentiation of species. The H. anthochroum strains are potential mycofumigation agents against postharvest diseases caused by F. oxysporum.

Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1.

G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1-it simultaneously increases agonist binding, decreases G--protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling.

A Novel Selective Inverse Agonist of the CB2 Receptor as a Radiolabeled Tool Compound for Kinetic Binding Studies.

The endocannabinoid system, and in particular the cannabinoid type 2 receptor (CB2R), raised the interest of many medicinal chemistry programs for its therapeutic relevance in several (patho)physiologic processes. However, the physico-chemical properties of tool compounds for CB2R (e.g., the radioligand [3H]CP55,940) are not optimal, despite the research efforts in developing effective drugs to target this system. At the same time, the importance of drug-target binding kinetics is growing since the kinetic binding profile of a ligand may provide important insights for the resulting in vivo efficacy. In this context we synthesized and characterized [3H]RO6957022, a highly selective CB2R inverse agonist, as a radiolabeled tool compound. In equilibrium and kinetic binding experiments [3H]RO6957022 showed high affinity for human CB2R with fast association (kon) and moderate dissociation (koff) kinetics. To demonstrate the robustness of [3H]RO6957022 binding, affinity studies were carried out for a wide range of CB2R reference ligands, spanning the range of full, partial, and inverse agonists. Finally, we used [3H]RO6957022 to study the kinetic binding profiles (i.e., kon and koff values) of selected synthetic and endogenous (i.e., 2-arachidonoylglycerol, anandamide, and noladin ether) CB2R ligands by competition association experiments. All tested ligands, and in particular the endocannabinoids, displayed distinct kinetic profiles, shedding more light on their mechanism of action and the importance of association rates in the determination of CB2R affinity. Altogether, this study shows that the use of a novel tool compound, i.e., [3H]RO6957022, can support the development of novel ligands with a repertoire of kinetic binding profiles for CB2R.

X-ray crystal structure of cytochrome P450 monooxygenase CYP101J2 from Sphingobium yanoikuyae strain B2.

The cytochrome P450 monooxygenases (P450s) catalyze a vast array of oxygenation reactions that can be useful in biocatalytic applications. CYP101J2 from Sphingobium yanoikuyae is a P450 that catalyzes the hydroxylation of 1,8-cineole. Here we report the crystallization and X-ray structure elucidation of recombinant CYP101J2 to 1.8 Å resolution. The CYP101J2 structure shows the canonical P450-fold and has an open conformation in the absence of substrate. Analysis of the structure revealed that CYP101J2, in the absence of substrate, forms a well-ordered substrate-binding channel that suggests a unique form of substrate guidance in comparison to other bacterial 1,8-cineole-hydroxylating P450 enzymes. Proteins 2017; 85:945-950. © 2016 Wiley Periodicals, Inc.

Four terpene synthases contribute to the generation of chemotypes in tea tree (Melaleuca alternifolia).

BACKGROUND: Terpene rich leaves are a characteristic of Myrtaceae. There is significant qualitative variation in the terpene profile of plants within a single species, which is observable as "chemotypes". Understanding the molecular basis of chemotypic variation will help explain how such variation is maintained in natural populations as well as allowing focussed breeding for those terpenes sought by industry. The leaves of the medicinal tea tree, Melaleuca alternifolia, are used to produce terpinen-4-ol rich tea tree oil, but there are six naturally occurring chemotypes; three cardinal chemotypes (dominated by terpinen-4-ol, terpinolene and 1,8-cineole, respectively) and three intermediates. It has been predicted that three distinct terpene synthases could be responsible for the maintenance of chemotypic variation in this species. RESULTS: We isolated and characterised the most abundant terpene synthases (TPSs) from the three cardinal chemotypes of M. alternifolia. Functional characterisation of these enzymes shows that they produce the dominant compounds in the foliar terpene profile of all six chemotypes. Using RNA-Seq, we investigated the expression of these and 24 additional putative terpene synthases in young leaves of all six chemotypes of M. alternifolia. CONCLUSIONS: Despite contributing to the variation patterns observed, variation in gene expression of the three TPS genes is not enough to explain all variation for the maintenance of chemotypes. Other candidate terpene synthases as well as other levels of regulation must also be involved. The results of this study provide novel insights into the complexity of terpene biosynthesis in natural populations of a non-model organism.

Apparent Affinity Estimates and Reversal of the Effects of Synthetic Cannabinoids AM-2201, CP-47,497, JWH-122, and JWH-250 by Rimonabant in Rhesus Monkeys.

Synthetic cannabinoids have been prohibited due to abuse liability and toxicity. Four such synthetic cannabinoids, AM-2201 ([1-(5-fluoropentyl)indol-3-yl]-naphthalen-1-ylmethanone), CP-47,497 (2-[(1R,3S)-3-hydroxycyclohexyl]-5-(2-methyloctan-2-yl)phenol), JWH-122 [(4-methylnaphthalen-1-yl)-(1-pentylindol-3-yl)methanone], and JWH-250 [2-(2-methoxyphenyl)-1-(1-pentylindol-3-yl)ethanone], were tested for their capacity to produce CB1 receptor-mediated discriminative stimulus effects in two groups of rhesus monkeys. One group (n = 4) discriminated Δ9-tetrahydrocannabinol (∆9-THC; 0.1 mg/kg i.v.), and a second group (n = 4) discriminated the cannabinoid antagonist rimonabant (1 mg/kg i.v.) while receiving 1 mg/kg/12 hours of ∆9-THC. AM-2201, JWH-122, CP-47,497, JWH-250, and ∆9-THC increased ∆9-THC lever responding. Duration of action was 1-2 hours for AM-2201, JWH-122, and JWH-250 and 4-5 hours for CP-47,497 and ∆9-THC. Rimonabant (1 mg/kg) surmountably antagonized the discriminative stimulus effects of all cannabinoid agonists; the magnitude of rightward shift was 10.6-fold for AM-2201, 10.7-fold for JWH-122, 11.0-fold for CP-47,497, and 15.7-fold for JWH-250. The respective pKB values were not significantly different: 6.61, 6.65, 6.66, and 6.83. In ∆9-THC-treated monkeys discriminating rimonabant, AM-2201 (0.1 and 0.32 mg/kg), JWH-122 (0.32 and 1 mg/kg), JWH-250 (1 and 3.2 mg/kg), and CP-47,497 (0.32, 1, and 3.2 mg/kg) produced not only rate-decreasing effects that were reversed by rimonabant, but also dose-dependent, rightward shifts in the rimonabant discrimination dose-effect function. These results show striking similarity in the CB1 receptor mechanism mediating the subjective effects of AM-2201, JWH-122, JWH-250, and CP-47,497. For products containing AM-2201 and JWH-122, a short duration of action could lead to more frequent use; moreover, inattention to differences in potency among synthetic cannabinoids could underlie unexpected toxicity. Rapid reversal of effects by intravenous rimonabant has potential value in emergency situations.

Genetic diversity and metabolic profile of Salvia officinalis populations: implications for advanced breeding strategies.

MAIN CONCLUSION: As a result of this work, we were able to characterize seven indigenous to Greece Salvia officinalis populations using genetic and metabolomic tools. These tools can be used to select the most promising genotypes, capable to design future breeding programs for high valuable varieties. An initial investigation was carried out to compare the genetic and metabolic diversity in S. officinalis grown in Greece and to discern the relationship between the two sets of data. Analysis of inter-simple sequence repeats (ISSR) revealed significant genetic differences among seven sage populations, which were grouped into three main clusters according to an UPGMA ISSR data-based dendrogram and Principle Coordinate Analysis. 80 loci were scored of which up to 90% were polymorphic at species level. According to the composition of their essential oil, the populations were classified into two chemotypes: 1.8 cineole/α-thujone and α-thujone/1.8 cineole. Additionally, a targeted ultra performance liquid chromatography (UPLC-MS/MS) method was used to qualify and quantify phenolic compounds in methanolic extracts of the seven sage genotypes according to which they were districted in six clusters among the sage populations. The main compounds characterizing the seven genotypes were rosmarinic acid and carnosol, followed by apigenin-7-O-glucoside (Ap7glc), and luteolin-7-O-glucoside (Lu7glc). The correlation between matrices obtained from ISSR data and metabolic profiles was non-significant. However, based on the differences in metabolic fingerprint, we aimed to define populations using as main selection criteria the high polyphenol content and desired essential oil composition, using state to the art analytical tools for the identification of parent lines for breeding programs.

The α-Terpineol to 1,8-Cineole Cyclization Reaction of Tobacco Terpene Synthases.

Flowers of Nicotiana species emit a characteristic blend including the cineole cassette monoterpenes. This set of terpenes is synthesized by multiproduct enzymes, with either 1,8-cineole or α-terpineol contributing most to the volatile spectrum, thus referring to cineole or terpineol synthase, respectively. To understand the molecular and structural requirements of the enzymes that favor the biochemical formation of α-terpineol and 1,8-cineole, site-directed mutagenesis, in silico modeling, and semiempiric calculations were performed. Our results indicate the formation of α-terpineol by a nucleophilic attack of water. During this attack, the α-terpinyl cation is stabilized by π-stacking with a tryptophan side chain (tryptophan-253). The hypothesized catalytic mechanism of α-terpineol-to-1,8-cineole conversion is initiated by a catalytic dyad (histidine-502 and glutamate-249), acting as a base, and a threonine (threonine-278) providing the subsequent rearrangement from terpineol to cineol by catalyzing the autoprotonation of (S)-(-)-α-terpineol, which is the favored enantiomer product of the recombinant enzymes. Furthermore, by site-directed mutagenesis, we were able to identify amino acids at positions 147, 148, and 266 that determine the different terpineol-cineole ratios in Nicotiana suaveolens cineole synthase and Nicotiana langsdorffii terpineol synthase. Since amino acid 266 is more than 10 Å away from the active site, an indirect effect of this amino acid exchange on the catalysis is discussed.

Fluoro-Carba-Sugars are Glycomimetic Activators of the glmS Ribozyme.

The glmS ribozyme is a bacterial gene-regulating riboswitch that controls cell wall synthesis, depending on glucosamine-6-phosphate as a cofactor. Due to the presence of this ribozyme in several human pathogen bacteria (e.g., MRSA, VRSA), the glmS ribozyme represents an attractive target for the development of artificial cofactors. The substitution of the ring oxygen in carbohydrates by functionalized methylene groups leads to a new generation of glycomimetics that exploits distinct interaction possibilities with their target structure in biological systems. Herein, we describe the synthesis of mono-fluoro-modified carba variants of α-d-glucosamine and ß-l-idosamine. (5aR)-Fluoro-carba-α-d-glucosamine-6-phosphate is a synthetic mimic of the natural ligand of the glmS ribozyme and is capable of effectively addressing its unique self-cleavage mechanism. However, in contrast to what was expected, the activity is significantly decreased compared to its non-fluorinated analog. By combining self-cleavage assays with the Bacillus subtilis and Staphylococcus aureus glmS ribozyme and molecular docking studies, we provide a structure-activity relationship for fluorinated carba-sugars.

Coupled reactions by coupled enzymes: alcohol to lactone cascade with alcohol dehydrogenase-cyclohexanone monooxygenase fusions.

The combination of redox enzymes for redox-neutral cascade reactions has received increasing appreciation. An example is the combination of an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO). The ADH can use NADP+ to oxidize cyclohexanol to form cyclohexanone and NADPH. Both products are then used by CHMO to produce ε-caprolactone. In this study, these two redox-complementary enzymes were fused, to create a self-sufficient bifunctional enzyme that can convert alcohols to esters or lactones. Three different ADH genes were fused to a gene coding for a thermostable CHMO, in both orientations (ADH-CHMO and CHMO-ADH). All six fusion enzymes could be produced and purified. For two of the three ADHs, we found a clear difference between the two orientations: one that showed the expected ADH activity, and one that showed low to no activity. The ADH activity of each fusion enzyme correlated with its oligomerization state. All fusions retained CHMO activity, and stability was hardly affected. The TbADH-TmCHMO fusion was selected to perform a cascade reaction, producing ε-caprolactone from cyclohexanol. By circumventing substrate and product inhibition, a > 99% conversion of 200 mM cyclohexanol could be achieved in 24 h, with > 13,000 turnovers per fusion enzyme molecule.

Acute and neuropathic orofacial antinociceptive effect of eucalyptol.

Terpenes have a wide range of pharmacological properties, including antinociceptive action. The anti-inflammatory and antinociceptive effects of eucalyptol are well established. The purpose of this study was to evaluate the antinociceptive effect of eucalyptol on acute and neuropathic orofacial pain in rodent models. Acute orofacial and corneal nociception was induced with formalin, capsaicin, glutamate and hypertonic saline in mice. In another series, animals were pretreated with capsazepine or ruthenium red to evaluate the involvement of TRPV1 receptors in the effect of eucalyptol. In a separate experiment, perinasal tissue levels of IL-1ß, TNF-α and IFN-γ were measured. Rats were pretreated with eucalyptol before induction of temporomandibular joint pain with formalin or mustard oil. In another experiment, rats were submitted to infraorbital nerve transection (IONX) to induce chronic pain, followed by induction of mechanical hypersensitivity using Von Frey hairs. Locomotor performance was evaluated with the open-field test, and molecular docking was conducted on the TRPV1 channel. Pretreatment with eucalyptol significantly reduced formalin-induced nociceptive behaviors in all mouse strains, but response was more homogenous in the Swiss strain. Eucalyptol produced antinociceptive effects in all tests. The effect was sensitive to capsazepine but not to ruthenium red. Moreover, eucalyptol significantly reduced IFN-γ levels. Matching the results of the experiment in vivo, the docking study indicated an interaction between eucalyptol and TRPV1. No locomotor activity changes were observed. Our study shows that eucalyptol may be a clinically relevant aid in the treatment of orofacial pain, possibly by acting as a TRPV1 channel antagonist.