Ferredoxin-dependent iron-sulfur flavoprotein glutamate synthase (GlsF) from the cyanobacterium Synechocystis sp PCC 6803: Expression and assembly inEscherichia coli

The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains two diff erent glutamate synthases whose genes, gltB and glsF (previously known as g ltS), have been cloned (F. Navarro et al., 1995, Plant Mol. Biol. 27, 753-7 67). The glsF gene has been expressed in the glutamate auxotrophic Escheric hia coli strain CLR207 RecA, but the corresponding protein does not complem ent the auxotrophy. The transformed strain showed ferredoxin-dependent glut amate 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 Cys 37 to form the mature enzyme in E. coli, as occurs with the large subunit o f its own NADPH-GOGAT. The recombinant Fd-GOGAT has been purified to electr ophoretic homogeneity, using as the main purification step a ferredoxin-aff inity chromatography. The pure enzyme, with a molecular mass of about 180 k Da, shows an absorption spectrum characteristic of iron-sulfur flavoprotein s. The analyses of the prosthetic groups indicate that Fd-GOGAT contains on ly one FMN, but no FAD, and one [Fe-3-4S](+,0) cluster per molecule. Oxidat ion-reduction titration, using absorbance changes of the FMN group in the v isible region, gave a midpoint redox potential of -200 +/- 25 mV at pH 7.5. The recombinant enzyme is strictly ferredoxin-dependent and shows apparent I, values similar to those of the native Synechocystis protein: 4.5 vs 3.5 mu M, 2.2 vs 2.5 mM, and 0.6 vs 0.5 mM for ferredoxin, glutamine, and 8-ox oglutarate, respectively. The addition of the reductant dithionite to the e nzyme resulted in the loss of the absorption peak at 436 nm, characteristic of oxidized flavins, which was restored by the anaerobic addition of 2-oxo glutarate, in the presence of glutamine. (C) 2000 Academic Press.