A new bacterial dehydrogenase oxidizing the lignin model compound guaiacylglycerol beta-O-4-guaiacyl ether

A lignin model compound, named in short guaiagylglycerol beta-guaiacyl ether (GGE), contains the beta-0-4 ether linkage that is common in the chemical structure of lignin. A Pseudomonas sp. (GU5) had been isolated as an organism able to grow with GGE as the sole source of carbon and energy. When grown on vanillate, the bacteria contained a NAD+ -dependent dehydrogenase converting GGE to a 355 nm absorbing product. The enzyme, named GGE-dehydrogenase, was purified about 160-fold using gel permeation, ion exchange on DEAE-Sephadex, and dye-ligand affinity chromatography. The new protein was about 52 kDa in apparent size with but one polypeptide chain after denaturation and reduction. According to several criteria, the product of GGE oxidation (Km = 12 microM) was identified as the corresponding conjugated ketone at the alpha-carbon of the C3 side-chain. The secondary alcohol function in GGE was apparently the sole target of the enzyme action. However the conversion of GGE into ketone catalyzed by the enzyme was only partial, and did not exceed 50%, probably because only one of the alpha-enantiomers was susceptible to enzyme attack. In contrast the ketone, either made by organic synthesis or by enzymic oxidation of GGE, could be totally reduced back to GGE (Km = 13 microM at pH 8.4, 8 microM at neutral pH), with NADH as the reductant, as confirmed by UV absorption and NMR spectra. Other model compounds with no primary alcoholic function, ether linkage or phenolic group were also substrates for the enzyme, confirming the specificity of GGE-dehydrogenase for the alpha-carbon position. Conjugation of the alpha-ketone with an adjacent phenolic nucleus interfered strongly with equilibrium constants and redox potentials of the system according to pH, and the enzyme displayed widely different optima with pH over 9 when oxidizing GGE, below 7 when reducing the ketone. Equilibrium studies showed that the ketone/GGE potential was -0.37 volt at pH 8.7, -0.23 volt at pH 7 (30 degrees C). The significance of this new dehydrogenase and its properties are discussed, especially in the general concern of lignin biodegradation.     

Cellular and biochemical indicators assessing the quality of a marine environment

Environmental stressors as well as the direct or combined effects of pollutants could be harmful to the populations living in a marine environment and the reproductive and nutritive processes could be impaired in a deteriorating environment. Sublethal effects of pollutants were studied in the blue mussel Mytilus edulis L., a good bioaccumulator of contaminants. Blue mussels of 3.5 cm were sampled on a rocky substrate at Pointe-Mitis (48° 40N, 68° 02W) along the coast of the St-Lawrence estuary. Mussels were placed in experimental tanks, fed, supplemented with mineral salts and continuous sea water flow and kept 72 h before the exposure to 0.01 µg l^–1 and 0.3 µg l^–1 methylmercury hydroxide in the presence or absence of selenium, at a concentration of 125 µg l^–1, a possible antagonist of methylmercury. The contamination protocol was performed during 45 days and a 14 day period of recuperation was allowed. The stress caused by the transplantation of mussels in the laboratory tanks and/or by the presence of pollutants was evaluated by a general indicator of stress developed in our laboratory, the measure of the lysosomal membrane fragility (LMF) of the digestive gland, according to the method developed by Moore (1976). The effects of contamination on metabolism were measured by the study of the variations of the malate dehydrogenase activity (MDH), a key enzyme of the aerobic metabolism. The first days of the contamination period led to an increased metabolism in the mantle and to a detoxifying mechanism in the hepatopancreas. At days 22 and 29 of the experiment, the affinity of the MDH was greatly decreased with both concentrations of methylmercury and selenium, suggesting a competitive inhibition of the enzymatic activity by the pollutants. LMF increased as the mussels were kept longer in the tanks. Methylmercury increased the stress undergone by the mussels. LMF gives information about the degree of stress of the organism while the biochemical indicator informs about the metabolic effects of sublethal concentrations of pollutants.     

Properties of γ-aminobutyraldehyde dehydrogenase from Escherichia coli

γ-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source. The enzyme has an M_r of 195 000±10 000 in its dimeric form with an M_r of 95 000±1000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity. K_m values are 31.3±6.8 μM and 53.8±7.4 μM for Δ-1-pyrroline and NAD⁺, respectively. An inhibitory capacity for NADH is also shown using the purified enzyme.     

The essential cationic charge of phospholipid polar head in the reactivation of -β-hydroxybutyrate apodehydrogenase revealed by cationic surfactants

Attempts to reactivate purified -β-hydroxybutyrate apodehydrogenase, a lecithin-requiring enzyme, have been carried out using neutral, anionic, cationic and zwitterionic surfactants. Cationic and zwitterionic compounds exclusively are able to partially replace phosphatidylcholine, the reactivating phospholipid. The extent of reactivation depends on the steric hindrance of the polar head and on the hydrophobic tail length. A molecule bearing a positive charge and an aliphatic chain is the sole structure absolutely required for activity. However the presence of a negative charge is important for enzyme binding to amphiphilic structures and for the efficiency of reactivation.     

Modification of redox equilibria between heme and flavin within yeast flavocytochrome b2 (l-lactate cytochrome c reductase) upon binding of pyruvate,the reaction product

Direct determinations of the concentration of semiquinone spin in redox equilibrium with the cytochrome b₂ moiety were carried out at room temperature in the presence of added pyruvate or in its absence. Results show that redox potentials of the one-electron couples of the prosthetic flavin are markedly affected by binding of pyruvate the reaction product in the oxidation of l-lactate. The proportion of flavin semiquinone nearly reaches then 100 per cent.     

Implication of glutamate, isocitrate and malate deshydrogenases in nitrogen assimilation in the cadmium-stressed tomato

Tomato seedlings grown on nitric medium and treated with various cadmium concentrations (0 to 50 microM) were used. Results obtained show that cadmium remains predominantly located in the roots, which then seem to play the role of trap-organs. Increasing cadmium concentration in the medium leads particularly to a decrease in NO3- accumulation, together with a decrease in the activity of glutamine synthetase and in the quantity of plastidic isoform ARNm (GS2), and, on the contrary, to an increase of the cytosolic isoform ARNm (GS1). On the other hand, stimulations were observed for NADH-dependent glutamate synthase, NADH-dependent glutamate dehydrogenase, ARNm quantity of this enzyme, ammonium accumulation, and protease activity. In parallel, stimulations were observed for NAD+ and NADP+-dependent malate dehydrogenase and NADP+-dependent isocitrate dehydrogenase. These results were discussed in relation to the hypothesis attributing to the dehydrogenase enzymes (GDH, MDH, ICDH) an important role in the plant defence processes against cadmium-induced stresses.