Ferredoxin-dependent iron-sulfur flavoprotein glutamate synthase (GlsF) from the Cyanobacterium synechocystis sp. PCC 6803: expression and assembly in Escherichia coli.
The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains two different glutamate synthases whose genes, gltB and glsF (previously known as gltS), have been cloned (F. Navarro et al., 1995, Plant Mol. Biol. 27, 753-767). The glsF gene has been expressed in the glutamate auxotrophic Escherichia coli strain CLR207 RecA, but the corresponding protein does not complement the auxotrophy. The transformed strain showed ferredoxin-dependent glutamate synthase (Fd-GOGAT) activity, demonstrating the capability of E. coli for providing and correctly assembling both the iron-sulfur center and the flavin cofactor of the enzyme. Fd-GOGAT (GlsF) is correctly cleaved at Cys37 to form the mature enzyme in E. coli, as occurs with the large subunit of its own NADPH-GOGAT. The recombinant Fd-GOGAT has been purified to electrophoretic homogeneity, using as the main purification step a ferredoxin-affinity chromatography. The pure enzyme, with a molecular mass of about 180 kDa, shows an absorption spectrum characteristic of iron-sulfur flavoproteins. The analyses of the prosthetic groups indicate that Fd-GOGAT contains only one FMN, but no FAD, and one [3Fe-4S](+,0) cluster per molecule. Oxidation-reduction titration, using absorbance changes of the FMN group in the visible region, gave a midpoint redox potential of -200 +/- 25 mV at pH 7.5. The recombinant enzyme is strictly ferredoxin-dependent and shows apparent K(M) values similar to those of the native Synechocystis protein: 4.5 vs 3.5 microM, 2.2 vs 2.5 mM, and 0.6 vs 0.5 mM for ferredoxin, glutamine, and 2-oxoglutarate, respectively. The addition of the reductant dithionite to the enzyme resulted in the loss of the absorption peak at 436 nm, characteristic of oxidized flavins, which was restored by the anaerobic addition of 2-oxoglutarate, in the presence of glutamine.
Navarro
F
F
Centro de Investigaciones Científicas Isla de la Cartuja, Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Avenida Américo Vespucio s/n, Sevilla, 41092, Spain.
Martín-Figueroa
E
E
Candau
P
P
Florencio
F J
FJ
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005415:Flavins / Q000032:analysis; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005816:Genetic Complementation Test; D018698:Glutamic Acid / Q000378:metabolism; D007501:Iron / Q000032:analysis; D007700:Kinetics; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013053:Spectrophotometry; D013379:Substrate Specificity; D013440:Sulfides / Q000032:analysis
D005288:Ferredoxins; D005415:Flavins; D005420:Flavoproteins; D011994:Recombinant Proteins; D013440:Sulfides; D018698:Glutamic Acid; D005486:Flavin Mononucleotide; D007501:Iron; D000594:Amino Acid Oxidoreductases; C042233:glutamate synthase (ferredoxin)