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Ferredoxin-dependent iron-sulfur flavoprotein glutamate synthase (GlsF) from the Cyanobacterium synechocystis sp. PCC 6803: expression and assembly in Escherichia coli.
Arch Biochem Biophys. 2000 Jul 15; 379(2):267-76.AB

Abstract

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.

Authors+Show Affiliations

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.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10898944

Citation

Navarro, F, et al. "Ferredoxin-dependent Iron-sulfur Flavoprotein Glutamate Synthase (GlsF) From the Cyanobacterium Synechocystis Sp. PCC 6803: Expression and Assembly in Escherichia Coli." Archives of Biochemistry and Biophysics, vol. 379, no. 2, 2000, pp. 267-76.
Navarro F, Martín-Figueroa E, Candau P, et al. Ferredoxin-dependent iron-sulfur flavoprotein glutamate synthase (GlsF) from the Cyanobacterium synechocystis sp. PCC 6803: expression and assembly in Escherichia coli. Arch Biochem Biophys. 2000;379(2):267-76.
Navarro, F., Martín-Figueroa, E., Candau, P., & Florencio, F. J. (2000). Ferredoxin-dependent iron-sulfur flavoprotein glutamate synthase (GlsF) from the Cyanobacterium synechocystis sp. PCC 6803: expression and assembly in Escherichia coli. Archives of Biochemistry and Biophysics, 379(2), 267-76.
Navarro F, et al. Ferredoxin-dependent Iron-sulfur Flavoprotein Glutamate Synthase (GlsF) From the Cyanobacterium Synechocystis Sp. PCC 6803: Expression and Assembly in Escherichia Coli. Arch Biochem Biophys. 2000 Jul 15;379(2):267-76. PubMed PMID: 10898944.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Ferredoxin-dependent iron-sulfur flavoprotein glutamate synthase (GlsF) from the Cyanobacterium synechocystis sp. PCC 6803: expression and assembly in Escherichia coli. AU - Navarro,F, AU - Martín-Figueroa,E, AU - Candau,P, AU - Florencio,F J, PY - 2000/7/19/pubmed PY - 2000/8/29/medline PY - 2000/7/19/entrez SP - 267 EP - 76 JF - Archives of biochemistry and biophysics JO - Arch Biochem Biophys VL - 379 IS - 2 N2 - 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. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/10898944/Ferredoxin_dependent_iron_sulfur_flavoprotein_glutamate_synthase__GlsF__from_the_Cyanobacterium_synechocystis_sp__PCC_6803:_expression_and_assembly_in_Escherichia_coli_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003986100918940 DB - PRIME DP - Unbound Medicine ER -