Francisco Galván

Purification and molecular properties of ferredoxin-glutamate synthase from Chlamydomonas reinhardii

Ferredoxin-glutamate synthase (EC 1.4.7.1) from Chlamydomonas reinhardii has been purified to electrophoretic homogeneity, with a specific activity of 10.4 units mg-1 protein, by a method which included chromatography on diethylaminoethyl sephacel and hydroxylapatite, and ferredoxin-sepharose affinity treatment. The enzyme is a single polypeptide chain of Mr 146000 dalton which shows an absorption spectrum with maxima at 278, 377 and 437 nm, and an A276/A437 absorptivity ratio of 7.0. The anaerobic addition of dithionite results in the loss of the absorption peak at 437 nm, which is restored upon reoxidation of the enzyme with an excess of 2-oxoglutarate, alone or in the presence of glutamine. This indicates the presence in the enzyme of a flavin prosthetic group, which is functional during the catalysis. The ferredoxin-glutamate synthase can be assayed with methyl viologen, chemically reduced with dithionite, but it is unable to use reduced pyridine nucleotide. Azaserine, 6-diazo-5-oxo-norleucine, bromocresol green and p-hydroxymercuribenzoate are potent inhibitors of this activity, which, on the other hand, is stable upon heating at 45°C for 10 min.

Purification and characterization of the NADH-glutamate synthase from Chlamydomonas reinhardii

Summary Chlamydomonas cells contain two enzymes with glutamate synthase (GOGAT) activity, which are specific, respectively, for reduced pyridine nucleotide (NADH) or reduced ferredoxin, as reductant. These enzymes, which may also use reduced methyl viologen as electron donor, can be separated by ion exchange chromatography on DEAE-sephacel. The NADH-GOGAT has been purified 350-fold by a method that uses affinity chromatography on blue-sepharose. The enzymatic complex keeps associated two additional activities which can be separately assayed; one is an NADH-diaphorase, that uses ferricyanide as electron acceptor, and the other is a reduced methyl viologen (MVH)-GOGAT, which cannot use benzyl viologen (BVH) or flavins as electron carrier. The NADH-GOGAT is specific for glutamine and 2-oxoglutarate as amido group donor and acceptor, respectively. Azaserine or 6-diazo-5-oxo-L-norleucine (DON), at 5 mM, inhibit the NADH- or MVH-GOGAT activity, but not the NADH-diaphorase activity of the complex. p -Hydroxymercuribenzoate ( p -HMB) strongly inhibits all the activities of the NADH-GOGAT complex, but the NADH-diaphorase is more resistant than the NADH-GOGAT.

STUDIES ON THE SUITABILITY OF ALGINATE-ENTRAPPED CHLAMYDOMONAS REINHARDTII CELLS FOR SUSTAINING NITRATE CONSUMPTION PROCESSES

Some aspects of the suitability of alginate beads entrapping Chlamydomonas reinhardtii cells for nitrate consumption from nitrate-containing waters were studied and discussed. Among 14 different metal cations tested as gel bead stabilizing agents, only calcium and barium formed beads showing nitrate-consuming activity. Pure calcium alginate cell entrapment resulted in the most suitable method for active cell immobilization compared to alginate-composite-gel beads based on poly-vinylcaprolactam (PVCL) and poly-vinylpyrrolidone (PVP). To perform a continuous nitrate consumption process, calcium alginate-entrapped cells were first grown in a 2.5 l airlift-loop reactor. A cell loading of about 150 microg Chl. g(-1) gel was achieved. Afterwards, five days nitrate consumption processes were performed and three different dilution rates were applied: (i) D < mu; (ii) D = mu; (iii) D > mu, where mu is the specific growth rate (h(-1)). The maximum consumption rates calculated for each dilution rate were: (i) 3.8, (ii) 6.4 and (iii) 7.2 mg nitrate mg(-1) Chl. h(-1). For low dilution rates (D < mu) some nitrite (< 300 microM) was excreted into the culture medium. However, this concentration of nitrite was not high enough to inhibit nitrate consumption.

Purification and properties of the siroheme-containing ferredoxin-nitrite reductase from Chlamydomonas reinhardtii

Abstract Assimilatory ferredoxin-nitrite reductase (ammonia: ferredoxin oxidoreductase, EC 1.7.7.1) from the green alga Chlamydomonas reinhardtii has been purified 340-fold with a specific activity of 89.0 U/mg protein. The enzyme has an M r of 86 000 and is composed for two subunits of M r 63 000 and 25000, respectively. Nitrite reductase shows absorption maxima at 280, 386 (Soret), 574 (α) and 690 nm, with an e 386 of 2.98 · 10 4 M −1 · cm −1 , and an absorbance ratio, A 280 / A 386 , of 1.76. Anaerobic addition of dithionite results in the loss of the 574 and 690 nm peaks, and the appearance of a shoulder at 590 nm. With nitrite, the 690 nm absorption band of substrate-free enzyme disappears and the absorbance in the α region is altered. CO inhibits nitrite reductase and forms a complex with the dithionite-reduced enzyme, with absorption maxima at 554 and 595 nm. These results suggest that ferrodoxin-nitrite reductase from C. reinhardtii contains siroheme as prosthetic group, which is involved in the catalytic reduction of nitrite. Analytical data based on the absorption of the pyridine hemochromogen derivative showed 0.65 molecules of siroheme per enzyme molecule

Glutamine Synthetase in Legumes: Recent Advances in Enzyme Structure and Functional Genomics

Glutamine synthetase (GS) is the key enzyme involved in the assimilation of ammonia derived either from nitrate reduction, N2 fixation, photorespiration or asparagine breakdown. A small gene family is encoding for different cytosolic (GS1) or plastidic (GS2) isoforms in legumes. We summarize here the recent advances carried out concerning the quaternary structure of GS, as well as the functional relationship existing between GS2 and processes such as nodulation, photorespiration and water stress, in this latter case by means of proline production. Functional genomic analysis using GS2-minus mutant reveals the key role of GS2 in the metabolic control of the plants and, more particularly, in carbon metabolism.

Ferredoxin-glutamate synthase from Chlamydomonas reinhardii. Prosthetic groups and preliminary studies of mechanism

1. 1. Ferredoxin-glutamate synthase from the green alga Chlamydomonas reinhardii appears to contain as prosthetic groups, 1 FAD, 1 FMN and 1 |3Fe-xS | cluster, per molecule of Mr = 146,000. 2. 2. The synthesis of glutamate, catalyzed by this enzyme, proceeds through the formation of an enzyme-bound free radical of flavin semiquinone. 3. 3. This enzyme may catalyze the ferredoxin-dependent reduction of 2-oxoglutarate, in the absence of glutamine.

Biological viability of Chlamydomonas reinhardtii cells entrapped in alginate beads for ammonium photoproduction

Abstract The green alga Chlamydomonas reinhardtii was immobilized by entrapment in Ca 2+ -alginate gel for ammonium photoproduction. The physical characteristics of the beads, their stability and ammonium-photoproduction capacity were affected by a variety of interactive factors, including cell loading, alginate viscosity and concentration, and the nature of the buffered medium. Electron micrographs show alterations in the morphology of the immobilized cells. Entrapped C. reinhardtii cells retained their biological viability, maintaining normal photosynthetic and respiratory activities, and they grew inside the gel beads, with a generation time of 9 h as compared with the 8 h shown by free cells under similar conditions. The unusually high rates for nitrite uptake and ammonium photoproduction, as well as the nitrite reductase activity shown by alginate-immobilized cells, were related to the changes in the membrane permeability induced by cell-matrix interaction.

Purification and physicochemical characterization of O-acetyl-L-serine sulfhydrylase from Chlamydomonas reinhardtii

Abstract O- Acetyl - l - serine sulfhydrylase (OASSase) from the green alga Chlamydomonas reinhardtii has been purified (spec. act. 41.15 units/mg) using hydrophobic chromatography on phenyl-Sepharose as basic step. The enzyme, Mr = 65 000, shows absorption maxima at 280 and 390 nm, indicative of the presence of pyridoxal-5′-phosphate as prosthetic group, and an A280/A390 absorptivity ratio of 6.2. Kinetic data indicate Km valued of 2 mM and 75 μM for O- acetyl - l - serine , respectively. l -Cyteine and l -Methionine are effective inhibitors of the OASSase activity, which is also inhibited by hydroxylamine and p-hydroxymercuribenzoate (pHMB).

Reassimilation of ammonium in Lotus japonicus

This review summarizes the most recent results obtained in the analysis of two important metabolic pathways involved in the release of internal sources of ammonium in the model legume Lotus japonicus: photorespiratory metabolism and asparagine breakdown mediated by aparaginase (NSE). The use of photorespiratory mutants deficient in plastidic glutamine synthetase (GS2) enabled us to investigate the transcriptomics and metabolomic changes associated with photorespiratory ammonium accumulation in this plant. The results obtained indicate the existence of a coordinate regulation of genes involved in photorespiratory metabolism. Other types of evidence illustrate the multiple interconnections existing among the photorespiratory pathway and other processes such as intermediate metabolism, nodule function, and secondary metabolism in this plant, all of which are substantially affected in GS2-deficient mutants because of the impairment of the photorespiratory cycle. Finally, the importance of asparagine metabolism in L. japonicus is highlighted because of the fact that asparagine constitutes the vast majority of the reduced nitrogen translocated between different organs of this plant. The different types of NSE enzymes and genes which are present in L. japonicus are described. There is a particular focus on the most abundant K(+)-dependent LjNSE1 isoform and how TILLING mutants were used to demonstrate by reverse genetics the importance of this particular isoform in plant growth and seed production.

Nitrate and ammonium assimilatory enzymes

Protocols for extraction and assay of the key enzymes of nitrate and ammonium assimilation in Lotus japonicus are given in this chapter, together with the peculiarities observed for these assays in different organs and physiological situations of the plant.