An oxidoreductase with an extremely broad substrate specificity reducing reversibly 2‐oxocar‐boxylates at the expense of reduced artificial redox mediators to (2R)‐hydroxycarboxylates has been purified to a specific activity of up to 1800 μmol · min−1· mg−1 for the reduction of phenylpyruvate. The membrane‐bound non‐pyridine nucleotide‐dependent enzyme appears in the form of various oligomers of the 80‐kDa monomer. The isoelectric point is 5.1. Based on a molecular mass of 80 kDa the enzyme contains up to one molybdenum, four iron and four sulphur atoms. After growth on 99Mo‐labelled molybdate, enzyme and radioactivity coincided as shown by gel electrophoresis. Permanganate oxidation delivers 0.7 mol pterin‐6‐carboxylic acid. The molybdenum cofactor is a mononucleotide. The enzyme is inhibited by cyanide. The first 20 amino acids have been determined. The enzyme belongs to the rare group of molybdoenzymes which possess no further prosthetic groups than the iron‐sulphur clusters.
During growth on L-cysteine ethylester, Chlorella fisca (211-8b) accumulated a substance which contained bound sulfide, which could be liberated by reduction with dithioerythritol (DTE) as inorganic sulfide.This substance was extracted with hot methanol and purified by thin layer chromatography. This substance liberated free sulide when incubated with mono-and dithiols, and thiocyanate was formed after heating with KCN. The isolated substance cochromatographed with authentic sulfur flower using different solvent systems for thin layer chromatography, high pressure liquid chromatography, and the identical spectrum with a relative Xmax at 263 nm was found. The chemical structre was confirmed by mass spectrometry showing a molecular weight of 256 m/ e for the S% configuration. No labeled elemental sulfur was detected when the cells were grown on [Sjsulfate and L-cysteine ethylester indicating the origin of elemental sulfur from L-cysteine ethylester. C. fusca seems to have enzymes for the metabolism of elemental sulfur, since it disappeared after prolonged growth into the stationary phase. Cysteine was formed from O-acetyl-L-serine and elemental sulfur in the presence of thiol groups and purified cysteine synthase from spinach or Chlorella.Phototrophic organisms form elemental sulfur (S°) under certain growth conditions. This was observed for Rhodospirillaceaelike Rhodopseudomonas and Rhodospirillum (13), Chromatiaceae-like Chromatium (12), and Chlorobiaceae-like Chlorobium (12). In blue green algae such as Oscillatoria (23) in 10-L bottles at light intensities of 7000 lux. For growth on radio-labeled sulfate carrier-free sulfate was added to the growth medium to reach a specific activity of 1725 cpm/nmol sulfate. Growth on [35S]sulfate was performed in 400-ml glass bottles connected to a Zn-acetate trap for labeled volatile sulfur compounds.Isolation of Bound Sulfide. Bound sulfide was isolated from cultures growing in 10-L bottles on sulfate and L-cysteine ethylester when the culture reached an optical density of 0.6 at 680 nm. Cells were harvested by centrifugation and washed once with distilled H20. Bound sulfide was extracted with hot methanol. After incubation in methanol at 80°C for 10 min, the cells were centrifuged for 10 min at 5000g and the pellet was extracted again with hot methanol. The combined supernatants were evaporated to drynes and resuspended in a small volume of petrolether. This crude extract was spotted on thin layer plates (Silica GF254, 0.5 mm) and developed with petrolether/diethylether (99.5/0.5) (solvent system 1). Bound sulfide with an RF of 0.84 was scraped off the plates, eluted with hot methanol, and spotted on a second plate which was developed with the solvent system 2 (acetone/water, 1/9) where bound sulfide stayed at the origin. The bound sulfide was eluted from the gel and spotted again on a TLC plate which was developed with ethylacetate (solvent system 3; RF = 0.86). The last plate was prerun with acetone and ethylacetate to remove impurities. This material was used ...
The green alga Chlorella fusca strain 21 1-8b (algal collection of Gottingen) is able to grow on more than 100 different sulphur sources such as mercaptides, disulphides, thioethers, sulphinic and sulphonic acids including 'Good buffers', sulphoxides, sulphones and sulphate esters. This suggests that green algae might be of importance for degradation of xenobiotics and natural compounds in the overall sulphur cycle. The data presented describe the growth of C. fusca on such sulphur compounds and the influence of these compounds on enzymes of the assimilatory sulphate reduction sequence. I N T R O D U C T I O NSulphur compounds are formed, released and degraded in nature as normal constituents of the environment in a series of reactions summarized as the sulphur cycle (Zehnder & Zinder, 1980). However, man-made sulphur compounds in the form of volatiles such as SO2 and H2S or bound forms of sulphur such as catalysts, tensides, organic solvents and drugs are discharged in increasing amounts to our environment (Kellog et al., 1972). These compounds cause severe trouble not only to plants (SOz toxicity, acid rain), but possibly also to the aquatic system. The effect of anthropogenic sulphur compounds on green algae has not been studied systematically, although a survey using the cyanobacterium Synechococcus (Anacystis nidulans) has appeared (Schmidt et al., 1982). Only limited information is available about sulphur-containing substances used for growth of higher plants (Brunold & Erismann, 1975;Ellis, 1969;Hart & Filner, 1969), algae (Krauss, 1984;Saito, 1976) and cyanobacteria (Schmidt et al., 1982). Furthermore these substances could interfere with metabolic pathways such as uptake systems or regulatory signals of sulphur and nitrogen assimilation (Reuveny & Filner, 1977;Zink, 1984). This study was initiated to obtain some information on the possible value of a green alga for the detoxification of sulphur compounds of either biotic or abiotic origin. METHODSOrganism. Chlorella fusca strain 21 1-8b of the algal collection of Gottingen, FRG, was cultured on a sulphurdeficient medium (SD medium). It contained @M): KH2P04, 500; Mg(N03), .6H20, 2000; KN03, 5000; KLHPO,, 500; Ca(NO,),, 250; H$O,, 23; NazMo04.2H20, 44; Mn(N03),.6H20, 6.3; ZnCl,, 0.37; Cu(N03), .H,O, 0.16; plus 1 ml l-' of Fe-EDTA complex (8.9 g FeCI, . 4 H 2 0 and 16.7 g EDTA.2H20 dissolved in 500 ml water and aerated overnight; the filtrate was used) and 1 ml I-' of trace elements (containing, per litre, 1.43 mg H3BO3, 10 mg NaMo0,.2H20, 50 mg ZnCl, and 38 mg Cu(N0J2.2H20).The sulphur compounds of Tables 1-4 were added to a final concentration of 0-3 mM whereas the sulphur control contained 0.2 mM Na,S04. The preparation and addition of the sulphur sources is described below. Pirson flasks containing 750 ml growth medium were inoculated with 6 x lo6 cells, equivalent to about 0-002 mg chlorophyll. Cultures were grown in a Kniese apparatus at 27 "C and 14000 lx using normal air as CO, source.
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