Biocatalysed halogenation of nucleobase analogues.

The synthesis of halogenated nucleosides and nucleobases is of interest due to their chemical and pharmacological applications. Herein, the enzymatic halogenation of nucleobases and analogues catalysed by microorganisms and by chloroperoxidase from Caldariomyces fumago has been studied. This latter enzyme catalysed the chlorination and bromination of indoline and uracil. Pseudomonas, Citrobacter, Aeromonas, Streptomyces, Xanthomonas, and Bacillus genera catalysed the chlorination and/or bromination of indole and indoline. Different products were obtained depending on the substrate, the biocatalyst and the halide used. In particular, 85% conversion from indole to 5-bromoindole was achieved using Streptomyces cetonii.

Chloroperoxidase-mediated transformation of highly halogenated monoaromatic compounds.

Peroxidase transformations of widely distributed pollutants, tetra- and penta-chlorinated phenols and anilines, were studied using different peroxidases. Chloroperoxidase from Caldariomyces fumago was able to transform tetra- and penta-chlorinated phenols and anilines, while horseradish peroxidase, lignin peroxidase from Phanerochaete chrysosporium and versatile peroxidase from Bjerkandera adusta were able only to transform the halogenated phenols. Chloroperoxidase showed a specific activity on pentachlorophenol two orders of magnitude higher than lignin peroxidase and horseradish peroxidase, and one order of magnitude higher than versatile peroxidase. The main product from peroxidase oxidation in all cases was a polymeric and insoluble material. The insolubilization of halogenated phenols and anilines permits their removal, reduces their bioavailability, and thus reduces their environmental impact. The other minor products from the enzymatic transformation of highly chlorinated compounds were determined by mass spectrometry. Tetrachloroquinone, dimers and trimers of halogenated compounds were also identified. Chloroperoxidase was able to halogenate tetrachloroaniline to form pentachloroaniline.

Intra-cellular storage, transport and exocytosis of halogenated compounds in marine red alga Laurencia obtusa.

The production of secondary metabolites in seaweed have been related to a capability to partition compounds into cellular specialized storage structures, like gland cells and the corps en cerise (CC) or cherry bodies. The possible mechanisms that bring these compounds to the thallus surface remain poorly understood. Therefore, the aim of this work is perform a characterization of the CC and determine the intra-cellular dynamics of halogenated compounds in Laurencia obtusa. The dynamics of CC and the mechanisms related to the intra-cellular transport of halogenated compounds were evaluated by using optical tweezers and time-lapse video microscopy. The CC were isolated and its elemental composition was characterized using X-ray microanalysis. The cellular distribution of halogenated compounds was also demonstrated by fluorescence microscopy. Three-dimensional reconstruction technique was used to provide a visualization of the structures that connect CC to cell periphery. As main findings, we confirmed that the halogenated compounds are mainly found in CC and also in vesicles distributed along the cytoplasm and within the chloroplasts. We demonstrated that CC is mechanically fixed to cell periphery by a stalk-like connection. A vesicle transport though membranous tubular connections was seen occurring from CC to cell wall region. We also demonstrated a process of cortical cell death event, resulting in degradation of CC. We suggested that the vesicle transportation along membranous tubular connections and cell death events are related to the mechanisms of halogenated compounds exudation to the thallus surface and consequently with defensive role against herbivores and fouling.

Effect of photon flux density and temperature on the production of halogenated monoterpenes by Plocamium cartilagineum (Plocamiaceae, Rhodophyta).

The effect of different photon flux densities (PFD) and temperatures on the relative growth rate (RGR) and the concentration of three halogenated monoterpenes in samples of Plocamium cartilagineum L.( Dixon), a marine alga (Rhodophyceae), were studied. The highest RGR (22.8 +/- 0.04 d(-1)) was obtained at 15 degreebC and 41 ,mol m(-2) s)(-1) of PFD and the lowest (18.0 +/- 0.12 d(-1)) was obtained at 18 degrees C and 120 micromol m(-2) s(-1). The different temperatures and light used in assays did not affect significantly the production of organic compounds. The production of mertensene and violacene was not affected significantly. However, compound 1 reached the highest concentration at 15 degrees C and 65 micromol m(-2) s(-1)). The relationship between growth and production of monoterpenes of P. cartilagineum and the effect of temperature and the PFD were analyzed.

Comparison between crab hepatopancreas and rat liver uroporphyrinogen decarboxylase.

We characterized Uroporphyrinogen decarboxylase (UroD) (E.C. 4.1.1.37) in hepatopancreas of the crab Chasmagnathus granulatus as a first step to establish this enzyme as a possible biomarker for environmental contamination. We performed a comparative study of crab UroD with the enzyme UroD present in Wistar rat liver, which is known as a useful indicator of intoxication by polyhalogenated aromatic hydrocarbons (PAHs). The final products were the same in crab and rat UroD: the remaining substrate (8-carboxyl-porphyrinogen), the final product Coproporphyrinogen (4-COOH) and intermediate compounds with 7-, 6- and 5-COOH. The elimination of the second carboxyl group seems to be the rate-limiting step in this multiple decarboxylation, because large amounts of 7-COOH porphyrinogen are accumulated. The V(max)/K(m) ratio was 100-fold higher for rat liver UroD than for crab hepatopancreas UroD, suggesting a higher efficiency of the rat enzyme. Optimum pH for enzyme activity was 7.2 and 6.8 for crab and rat, respectively. Although both systems showed the same optimum temperature (47 degrees C), the activation energy was clearly different, 51.5 kJ/mol for C. granulatus and 5.4 kJ/mol for Rattus norvegicus (Wistar strain). Superdex 75 gel chromatography yielded a single symmetrical peak with an apparent molecular mass of 48+/-3 kDa for crab hepatopancreas UroD, suggesting the existence of only one enzymatic species in C. granulatus.