Insight into Biochemical Characterization of Plant Sesquiterpene Synthases.

A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with reduced reagent usage, it allows further reduction in the use of radioactive isotopes and flammable organic solvents. The sesquiterpene synthases previously characterized were expressed in yeast, and the plant-derived Thapsia garganica kunzeaol synthase TgTPS2 was tested in this method. KM for TgTPS2 was found to be 0.55 µM; the turnover number, kcat, was found to be 0.29 s-1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/KM was found to be 0.53 s-1 µM-1 for TgTPS2. The kinetic parameters were in agreement with previously published data.

Role of the Colletotrichum acutatum sesquiterpene synthase CaTPS in the biosynthesis of sesquiterpenoids.

Colletotrichum acutatum is a major fungal pathogen of fruit crops, which causes severe yield losses in strawberry production. A potential key factor in plant-pathogen interactions is fungal sesquiterpenoids which have mycotoxic and phytotoxic activities. The first committed step in sesquiterpenoid biosynthesis is performed by sesquiterpene synthases (TPS). Only a few TPSs have been functionally characterized from filamentous fungi and none from the genus Colletotrichum. Despite being an important fungal pathogen to agriculture, it is poorly understood at the molecular and chemical levels. The terpenoid biochemistry in Coll. acutatum strain SA 0-1 was studied and one Coll. acutatum TPS (CaTPS) was successfully cloned and characterized in yeast. CaTPS catalyses the biosynthesis of multiple sesquiterpenoids. The two major products are ß-caryophyllene and an unidentified sesquiterpenoid along with α-humulene as one of the minor sesquiterpenoid products. These products were also secreted by the fungus in strawberry fruit medium along with several other sesquiterpenoids indicating other TPSs are active during in vitro growth. ß-Caryophyllene and α-humulene are known cytotoxic products important for ecological interactions and are produced by SA 0-1. Interestingly, a gene expression analysis using quantitative real-time PCR revealed a significant increase in expression of CaTPS during strawberry fruit infection, thus indicating that it could be involved in fruit infection. This is, we believe, the first characterization of TPS in Colletotrichum spp. and terpenoid profiles of Coll. acutatum, which could facilitate studies on the role of terpenoids in the ecology of Coll. acutatum.

Thapsigargin--from Thapsia L. to mipsagargin.

The sesquiterpene lactone thapsigargin is found in the plant Thapsia garganica L., and is one of the major constituents of the roots and fruits of this Mediterranean species. In 1978, the first pharmacological effects of thapsigargin were established and the full structure was elucidated in 1985. Shortly after, the overall mechanism of the Sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibition that leads to apoptosis was discovered. Thapsigargin has a potent antagonistic effect on the SERCA and is widely used to study Ca2+-signaling. The effect on SERCA has also been utilized in the treatment of solid tumors. A prodrug has been designed to target the blood vessels of cancer cells; the death of these blood vessels then leads to tumor necrosis. The first clinical trials of this drug were initiated in 2008, and the potent drug is expected to enter the market in the near future under the generic name Mipsagargin (G-202). This review will describe the discovery of the new drug, the on-going elucidation of the biosynthesis of thapsigargin in the plant and attempts to supply the global market with a novel potent anti-cancer drug.

Identification and characterization of a kunzeaol synthase from Thapsia garganica: implications for the biosynthesis of the pharmaceutical thapsigargin.

Thapsigargin is a major terpenoid constituent of Thapsia garganica root. Owing to its potent antagonistic effect on the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, thapsigargin has been widely used to study Ca2+ signalling and is also a potential drug for prostate cancer. Despite its importance, thapsigargin biosynthesis in T. garganica remains unknown. In order to decipher thapsigargin biosynthesis, deep transcript sequencing (454 and Illumina) of the T. garganica root was performed, and two terpene synthases (TgTPS1/2) were identified. Functional characterization of their encoded enzymes in a metabolically engineered yeast revealed that TgTPS1 synthesized δ-cadinene, whereas TgTPS2 produced ten distinct terpenoids. However, cultivation of the TgTPS2-expressing yeast in pH-maintained conditions (pH 6-7) yielded one major oxygenated sesquiterpenoid, suggesting that formation of multiple terpenoids was caused by acidity. The major terpene product from TgTPS2 was identified as 6ß-hydroxygermacra-1(10),4-diene (kunzeaol) by mass-fragmentation pattern, retention index, the nature of its acid-induced degradation and NMR. Also, recombinant TgTPS2 efficiently catalysed the synthesis of kunzeaol in vitro from farnesyl diphosphate with a Km of 2.6 µM and a kcat of 0.03 s-1. The present paper is the first report of a kunzeaol synthase, and a mechanism for the transformation of kunzeaol into the thapsigargin backbone is proposed.

Several domains from VAR2CSA can induce Plasmodium falciparum adhesion-blocking antibodies.

BACKGROUND: Malaria caused by Plasmodium falciparum can result in several different syndromes with severe clinical consequences for the about 200 million individuals infected each year. During pregnancy, women living in endemic areas become susceptible to malaria due to lack of antibodies against a unique P. falciparum membrane protein, named VAR2CSA. This antigen is not expressed in childhood infections, since it binds chondroitin sulphate A (CSA) expressed on the intervillous space in the placenta. A vaccine appears possible because women acquire protective antibodies hindering sequestration in the placenta as a function of parity. A challenge for vaccine development is to design small constructs of this large antigen, which can induce broadly protective antibodies. It has previously been shown that one domain of VAR2CSA, DBL4-FCR3, induces parasite adhesion-blocking antibodies. In this study, it is demonstrated that other domains of VAR2CSA also can induce antibodies with inhibitory activity. METHODS: All VAR2CSA domains from the 3D7 and HB3 parasites were produced in Baculovirus-transfected insect cells. Groups of three rats per protein were immunized and anti-sera were tested for surface reactivity against infected erythrocytes expressing FCR3 VAR2CSA and for the ability to inhibit FCR3CSA parasite adhesion to CSA. The fine specificity of the immune sera was analysed by VAR2CSA peptide arrays. RESULTS: Inhibitory antibodies were induced by immunization with DBL3-HB3 T1 and DBL1-3D7. However, unlike the previously characterised DBL4-FCR3 response the inhibitory response against DBL1-3D7 and DBL3-HB3 T1 was poorly reproduced in the second rounds of immunizations. CONCLUSION: It is possible to induce parasite adhesion-blocking antibodies when immunizing with a number of different VAR2CSA domains. This indicates that the CSA binding site in VAR2CSA is comprised of epitopes from different domains.