Linalool Dehydratase-Isomerase,a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme ∼1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mm DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V_max) were 140 nanokatals mg⁻¹ for the linalool dehydratase and 410 nanokatals mg⁻¹ for the geraniol isomerase, with apparent K_m values of 750 μm and 500 μm, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2.1.X).     

Pine monoterpenes and pine bark beetles: a marriage of convenience for defense and chemical communication

Pine-feeding bark beetles (Coleoptera: Scolytidae) interact chemically with their host pines (Coniferales: Pinaceae) via the behavioral, physiological, and biochemical effects of one class of isoprenoids, the monoterpenes and their derivatives. Pine monoterpenes occur in the oleoresin and function as behaviorally active kairomones for pine bark beetles and their predators, presenting a classic example of tri-trophic chemical communication. The monoterpenes are also essential co-attractants for pine bark beetle aggregation pheromones. Ironically, pine monoterpenes are also toxic physiologically to bark beetles at high vapor concentrations and are considered an important component of the defense of pines. Research over the last 30 years has demonstrated that some bark beetle aggregation pheromones arise through oxygenation of monoterpenes, linking pheromone biosynthesis to the host pines. Over the last 10 years, however, several frequently occurring oxygenated monoterpene pheromone components (e.g., ipsenol, ipsdienol and frontalin) have also been shown to arise through highly regulated de novo pathways in the beetles (reviewed in Seybold and Tittiger, 2003). The most interesting nexus between these insects and their plant hosts involves the late-stage reactions in the monoterpenoid biosynthetic pathway, during which isomeric dimethylallyl diphosphate and isopentenyl diphosphate are ultimately elaborated to stereospecific monoterpenes in the trees and to hydroxylated monoterpenes or bicyclic acetals in the insects. There is signal stereospecificity in both production of and response to the monoterpenoid aggregation pheromones of bark beetles and in response to␣the monoterpenes of the pines. In the California fivespined ips, Ips paraconfusus, we have discovered a number of cytochome P450 genes that have expression patterns indicating that they may be involved in detoxifying monoterpene secondary metabolites and/or biosynthesizing pheromone components. Both processes result in the production of oxygenated monoterpenes, likely with varying degrees of stereospecificity. A behavioral analysis of the stereospecific response of I. paraconfusus to its pheromone is providing new insights into the development of an efficacious bait for the detection of this polyphagous insect in areas outside the western United States. In contrast, a Eurasian species that has arrived in California, the Mediterranean pine engraver, Orthotomicus (Ips) erosus, utilizes both a monoterpenoid (ipsdienol) and a hemiterpenoid (2-methyl-3-buten-2-ol) in its pheromone blend. The stereospecificity of the response of O. erosus to the monoterpenoid appears to be the key factor to the improved potency of the attractant bait for this invasive species.Dedicated to Professor David L. Wood on the occasion of his 75th birthday, January 8, 2006     

Adaptation to β‐myrcene catabolism in Pseudomonas sp. M1: An expression proteomics analysis

β‐Myrcene, a monoterpene widely used as a fragrance and flavoring additive, also possesses analgesic, anti‐mutagenic, and tyrosinase inhibitory properties. In order to get insights into the molecular mechanisms underlying the ability of Pseudomonas sp. M1 to catabolize β‐myrcene, an expression proteomics approach was used in this study. Results indicate that the catabolic enzyme machinery for β‐myrcene utilization (MyrB, MyrC, and MyrD and other uncharacterized proteins) is strongly induced when β‐myrcene is present in the growth medium. Since an M1 mutant, lacking a functional 2‐methylisocitrate dehydratase, is not able to grow in mineral medium with β‐myrcene or propionic acid as the sole C‐source, and also based on the expression proteomic analysis carried out in this study, it is suggested that the β‐myrcene catabolic intermediate propionyl‐CoA is channeled into the central metabolism via the 2‐methylcitrate cycle. Results also suggest that the major alteration occurring in the central carbon metabolism of cells growing in β‐myrcene‐containing media is related with the redistribution of the metabolic fluxes leading to increased oxaloacetate production. Other up‐regulated proteins are believed to prevent protein misfolding and aggregation or to play important structural roles, contributing to the adaptive alteration of cell wall and membrane organization and integrity, which are essential features to allow the bacterium to cope with the highly lipophilic β‐myrcene as C‐source.     

New cytotoxic-antineoplastic prenyl-1,2-naphthohydroquinone derivatives

Several new prenylnaphthohydroquinone derivatives have been prepared through the Diels–Alder condensation between -myrcene and 1,2-benzoquinone and evaluated for their cytotoxic activity against A-549, HT-29 and MB-231 cultured cell lines. All of them have shown GI₅₀ values in the μM level.     

Effect of Growth Stage on Essential‐Oil Yield and Composition of Daucus sahariensis

The essential oils obtained by hydrodistillation from Daucus sahariensis Murb . harvested at three different growth stages were characterized by GC/MS analysis. In total, 88 compounds were identified, with myristicin (29.8–51.7%), myrcene (6.7–31.1%), α‐pinene (11.6–14.8%), and limonene (5.3–11.5%) as main constituents. Monoterpene hydrocarbons were the most represented compounds in the oils of the plant samples collected during the flower‐budding and full‐flowering periods. On the contrary, during the fruiting stage, the oils were dominated by phenylpropanoids. The essential oils were subject of considerable variation in their composition during the various developmental stages, particularly concerning the content of myrcene that decreased significantly passing from the vegetative to the fruiting stage. Conversely, for myristicin, the opposite trend was observed. Furthermore, the essential‐oil yields were quite low during the flower‐budding phase (0.27%), but rapidly increased during plant development (0.63 and 0.68% for the flowering and fruiting phases, resp.).     

Volatiles of Curcuma mangga Val. & Zijp (Zingiberaceae) from Malaysia

Analysis by GC and GC/MS of the essential oil obtained from Malaysian Curcuma mangga Val. & Zijp (Zingiberaceae) rhizomes allowed the identification of 97 constituents, comprising 89.5% of the total oil composition. The major compounds were identified as myrcene (1; 46.5%) and β-pinene (2; 14.6%). The chemical composition of this and additional 13 oils obtained from selected Curcuma L. taxa were compared using multivariate statistical analyses (agglomerative hierarchical cluster analysis and principal component analysis). The results of the statistical analyses of this particular data set pointed out that 1 could be potentially used as a valuable infrageneric chemotaxonomical marker for C. mangga. Moreover, it seems that C. mangga, C. xanthorrhiza Roxb., and C. longa L. are, with respect to the volatile secondary metabolites, closely related. In addition, comparison of the essential oil profiles revealed a potential influence of the environmental (geographical) factors, alongside with the genetic ones, on the production of volatile secondary metabolites in Curcuma taxa.     

In situ recovery of the aroma compound perillene from stirred-tank cultured Pleurotus ostreatus using gas stripping and adsorption on polystyrene

Supplementation of the key metabolite, alpha-(Z)-acaridiol, to stirred-tank cultured Pleurotus ostreatus was used to demonstrate that integrated in situ product recovery resulted in high conversion rates and quantitative separation of the target product perillene from the nutrient medium. The conversion of beta-myrcene by P. ostreatus was scaled-up from shake-flasks into a controlled, stirred tank bioreactor equipped with gas stripping and adsorption on a polystyrene fixed bed. The formation of the attractive flavour compound perillene was measured daily using standard controlled capillary gas chromatography. The formation of alpha-(Z)-acaridiol was the metabolic bottleneck of the conversion of beta-myrcene to perillene. Efficient in situ recovery of the volatile product enabled quantitative separation of the pure flavour compound. Appropriated bioprocessing, i.e. in situ separation of product, steadily shifted the metabolic equilibria and thus accomplished high conversion rate and pure product.     

Influence of myrcene on inflammation,matrix accumulation in the kidney tissues of streptozotocin-induced diabetic rat

There is only limited literature studies on the activities of inflammation and matrix accumulation in the renal tissues of rats induced with diabetes through Streptozotocin. The present the investigation involves the examination of the protective actions of Myrcene (MYN), a monoterpene on the oxidative stress, inflammation, and matrix accumulation. For this purpose an experimental setup was created which involves injecting MYN 50 mg/kg for about 45 days in the STZ diabetic rats. Modifications in the enzymes, collagens, growth factor B₁ and Kappa factor P65 were identified and tracked. The levels of the inflammatory markers like TF-α1, ICAM-1, VCAM-1, MCP-1 were tracked and noted. The current experimental results showed an alteration in the glucose metabolism and enhanced condition. Also an increased level of TGF-β-1 and Nuclear factor-kB expression was seen in the renal tissues. MYN was found to reduce glucose oxidative stress and exhibit an anti-inflammatory effect via inhibiting NF-kB signalling. The conclusion of the current study reveals that MYN regulates the inflammatory activities and matrix accumulation by inhibiting the activities of inflammatory cytokine, pro-inflammatory signalling.