Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from plectranthus amboinicus

Linalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase

Diffusion of tin from TEC-8 conductive glass into mesoporous titanium dioxide in dye sensitized solar cells

The photoanode of a dye sensitized solar cell is typically a mesoporous titanium dioxide thin film adhered to a conductive glass plate. In the case of TEC-8 glass, an approximately 500 nm film of tin oxide provides the conductivity of this substrate. During the calcining step of photoanode fabrication, tin diffuses into the titanium dioxide layer. Scanning Electron Microscopy and Electron Dispersion Microscopy are used to analyze quantitatively the diffusion of tin through the photoanode. At temperatures (400 to 600 °C) and times (30 to 90 min) typically employed in the calcinations of titanium dioxide layers for dye sensitized solar cells, tin is observed to diffuse through several micrometers of the photoanode. The transport of tin is reasonably described using Fick\u27s Law of Diffusion through a semi-infinite medium with a fixed tin concentration at the interface. Numerical modeling allows for extraction of mass transport parameters that will be important in assessing the degree to which tin diffusion influences the performance of dye sensitized solar cells

Sewage sludge heavy metal analysis and agricultural prospects for Fiji

Insoluble residues produced in Waste Water Treatment Plants (WWTP) as by products are known as sewage sludge (SS). Land application of SS, particularly in agricultural lands, is becoming an alternative disposal method in Fiji. However, currently there is no legislative framework governing its use. SS together with its high nutrient and organic matter contents, constitutes some undesired pollutants such as heavy metals, which may limit its extensive use. The focus of this study therefore was to determine the total concentrations of Pb, Zn, Cd, Cu, Cr, Ni and Mn in the SS produced at the Kinoya WWTP (Fiji) and in the non-fertile soil amended with the SS at 20, 40, 60, 80% application rates and in the control (100% Soil). The bioavailable heavy metals were also determined as it depicts the true extent of metal contamination. The treatment mixtures were then used to cultivate cabbage plants in which the total heavy metal uptake was investigated. Total Zn (695.6 mg/kg) was present in the highest amounts in the 100% SS (control), followed by Pb (370.9 mg/kg), Mn (35.0 mg/kg), Cu (65.5 mg/kg), Cr (20.5 mg/kg) and finally Cd (13.5 mg/kg) and hence a similar trend was seen in all treatment mixtures. The potential mobility of sludgeborne heavy metals can be classified as Ni > Cu > Cd > Zn > Mn > Cr > Pb. Total metal uptake in plant leaves and stems showed only the bioavailable metals Cu, Cd, Zn and Mn, with maximum uptake occurring in the leaves. Ni, despite being highly mobile was not detected, due to minute concentrations in the SS treatments. Optimum growth occurred in the 20 and 40% SS treatments. However maximum Cu and Mn uptake occurred in the 40% SS treatment thereby making the 20% treatment the most feasible. Furthermore the total and bioavailable metal concentrations observed were within the safe and permitted limits of the EEC and USEPA legislations

Cholinesterase Research

This collection of 10 papers includes original as well as review articles focused on the cholinesterase structural aspects, drug design and development of novel cholinesterase ligands, but also contains papers focused on the natural compounds and their effect on the cholinergic system and unexplored effects of donepezil

Investigation of the function of delta-cadinene synthase with aza-analogues and site directedmutagenesis

Terpenes are one of the most structurally varied families of natural products with extraordinary chemical properties that have been exploited for numerous applications. Sesquiterpene synthases are a family of metal-dependent enzymes that catalyse the cyclisation of farnesyl diphosphate (FDP) into a myriad of complex C15-isoprenoid hydrocarbons, the sesquiterpenes. δ-Cadinene synthase (DCS) from Gossypium arboreum (cotton tree) catalyses the formation of δ-cadinene (DCN), a bicyclic intermediate in the biosynthesis of important phytoalexins such us gossypol. Two mechanistic proposals have been made for the formation of δ-cadinene: a 1,10-ring closure mechanism leading to the key intermediate germacradienyl cation, or a 1,6-ring closure leading to thealpha-bisabolyl carbocation. Previous investigation with fluorinated FDP analogues were in partial agreement with both scenarios and hence it was not possible to distinguish unambiguously between the two possible cyclisation reactions. To investigate the catalytic mechanism of DCS, enantiopure samples of the azaanalogues of alpha-bisabolyl cation and germacradienyl cation were needed. These compounds are designed as stable structural and electrostatic mimics of the putative short-lived carbocationic intermediates generated by terpene synthases, and hence often act as potent reversible competitive inhibitors (low Ki) of these enzymes. Here, the enantioselective total synthesis of R- and S- aza-analogues of the alpha-bisabolyl cation are described as well as the partial racemic synthesis of azagermacradienyl cation. Both enantiomers of aza-bisabolyl cation were goodmimics of α-bisabolene. They were competitive inhibitors of DCS, providing evidence for a 1,6-cyclisation closure. The second part of the project involved the investigation of the role of tryptophan 279 for the desolvation of the active site of DCS and therefore for the formation of DCN. Seven mutants of W279 were created. The data obtained showed that W279 is essential to prevent water from entering the active site and form the hydroxylate terpenoid germacradien-4-ol (GD4ol). Mutagenesis studies yielded a mutant, W279A, capable of making GD4ol as the sole product

Investigation of the function of delta-cadinene synthase with aza-analogues and site directedmutagenesis

Terpenes are one of the most structurally varied families of natural products with extraordinary chemical properties that have been exploited for numerous applications. Sesquiterpene synthases are a family of metal-dependent enzymes that catalyse the cyclisation of farnesyl diphosphate (FDP) into a myriad of complex C15-isoprenoid hydrocarbons, the sesquiterpenes. δ-Cadinene synthase (DCS) from Gossypium arboreum (cotton tree) catalyses the formation of δ-cadinene (DCN), a bicyclic intermediate in the biosynthesis of important phytoalexins such us gossypol. Two mechanistic proposals have been made for the formation of δ-cadinene: a 1,10-ring closure mechanism leading to the key intermediate germacradienyl cation, or a 1,6-ring closure leading to thealpha-bisabolyl carbocation. Previous investigation with fluorinated FDP analogues were in partial agreement with both scenarios and hence it was not possible to distinguish unambiguously between the two possible cyclisation reactions. To investigate the catalytic mechanism of DCS, enantiopure samples of the azaanalogues of alpha-bisabolyl cation and germacradienyl cation were needed. These compounds are designed as stable structural and electrostatic mimics of the putative short-lived carbocationic intermediates generated by terpene synthases, and hence often act as potent reversible competitive inhibitors (low Ki) of these enzymes. Here, the enantioselective total synthesis of R- and S- aza-analogues of the alpha-bisabolyl cation are described as well as the partial racemic synthesis of azagermacradienyl cation. Both enantiomers of aza-bisabolyl cation were goodmimics of α-bisabolene. They were competitive inhibitors of DCS, providing evidence for a 1,6-cyclisation closure. The second part of the project involved the investigation of the role of tryptophan 279 for the desolvation of the active site of DCS and therefore for the formation of DCN. Seven mutants of W279 were created. The data obtained showed that W279 is essential to prevent water from entering the active site and form the hydroxylate terpenoid germacradien-4-ol (GD4ol). Mutagenesis studies yielded a mutant, W279A, capable of making GD4ol as the sole product

36th Rocky Mountain Conference on Analytical Chemistry

Program, abstracts, and information about the 36th annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-sponsored by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Denver, Colorado, July 31 - August 5, 1994