Due to climate change, many lakes in Europe will be subject to higher variability of hydrological characteristics in their littoral zones. These different hydrological regimes might affect the use of allochthonous and autochthonous carbon sources. We used sandy sediment microcosms to examine the effects of different hydrological regimes (wet, desiccating, and wet-desiccation cycles) on carbon turnover. 13C-labelled particulate organic carbon was used to trace and estimate carbon uptake into bacterial biomass (via phospholipid fatty acids) and respiration. Microbial community changes were monitored by combining DNA- and RNA-based real-time PCR quantification and terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA. The shifting hydrological regimes in the sediment primarily caused two linked microbial effects: changes in the use of available organic carbon and community composition changes. Drying sediments yielded the highest CO2 emission rates, whereas hydrological shifts increased the uptake of allochthonous organic carbon for respiration. T-RFLP patterns demonstrated that only the most extreme hydrological changes induced a significant shift in the active and total bacterial communities. As current scenarios of climate change predict an increase of drought events, frequent variations of the hydrological regimes of many lake littoral zones in central Europe are anticipated. Based on the results of our study, this phenomenon may increase the intensity and amplitude in rates of allochthonous organic carbon uptake and CO2 emissions.
The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil fromThymussp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase fromT. vulgaris(TvTPS), theThymusspecies that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to γ-terpinene. The crystal structure of recombinantly expressed and purifiedTvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site ofTvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase fromSalviasp. and to (4S)-limonene synthase fromMentha spicata.
Background: Progesterone 5β-reductases (P5ßRs) catalyse one step in the stereospecific biosynthesis of cardenolides (potent repellents of herbivores and pharmaceutical stimulants of disordered heart muscle cells). They were originally discovered in the genus Digitalis and have since been frequently isolated from other angiosperms. Recombinant P5ßRs engineered in Escherichia coli host cells convert a broad spectrum of compounds in vitro, sometimes with higher efficiency than with progesterone. This observation suggests additional functions for cardenolide metabolism and promises future use in sustainable chemistry and biotechnology. Methods: A tissue complementary DNA (cDNA) library was screened for orthologous P5ßRs. Candidates were subcloned into expression vectors and overexpressed in E. coli cells. The recombinant P5ßR protein was investigated for catalytic activity with several related substrates. Using spectrophotometric assays, the biochemical parameters of the enzyme were calculated. A 3D model was created and was compared to the previously published P5ßR structure of Digitalis lanata and other plant P5ßR models.Results: Performing protein similarity searches in public databases and comparison of 3D protein structure models revealed four cDNA clones in a tissue library of Picea sitchensis (Bong.) Carrière putatively encoding P5ßRs. Succeeding with the expression of one clone in E. coli, the highly purified protein was unambiguously able to enantioselectively convert progesterone into 5ß-pregnane-3,20-dione. However, the catalytic activity to reduce the small molecule 2-cyclohexen-1-one was nearly 100 times faster. Methyl vinyl ketone was reduced similar to results from previously studied angiosperm resources.Conclusions: (i) The low catalytic efficiency for progesterone conversion agrees with the fact that conifers have not been reported to accumulate cardenolides. This finding suggests that alternate metabolic processes occur whereby the newly detected enzymes could transform smaller molecules rather than large ones such as progesterone.(ii) An ancient P5ßR gene appears to have existed in the last common ancestor of seed plants approximately 300 million years ago. If the diversification of P5ßRs, including the currently detected homologous iridoid synthase activity, was related to stress encountered during the transition to growth on land, then investigation of P5ßRs from pteridophytes and bryophytes should improve our knowledge of this enzyme class and elucidate the direction of evolution.
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