DOI: 10.1111/j.1432-1033.1992.tb16902.x. Purification and characterization of the ferredoxin‐glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301

Purification and characterization of the ferredoxin‐glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301

10.1111/j.1432-1033.1992.tb16902.x

Crossref journal-article

Wiley

European Journal of Biochemistry (311)

Abstract

Ferredoxin‐glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301 has been purified using, as main steps, ethanol fractionation in the presence of high ionic strength, ion‐exchange chromatography and ferredoxin‐Sepharose affinity chromatography. The overall process yielded an homogeneous enzyme with a specific activity of 30 U/mg protein, after a purification of 2800‐fold with a recovery of 43%. The molecular mass of the native protein was 156 kDa, as calculated from its Stokes radius (rs, 4.32 nm) and sedimentation coeficient (s20,w, 8.46 S). The size was also estimated by SDS/PAGE as 160 kDa, indicating that the native protein was a monomer. The enzyme exhibited absorption maxima at 279, 370 and 438 nm and a A279/A438 absorbance ratio of 11. One molecule of FMN, but not FAD, was found/molecule native protein. The addition of dithionite resulted in the loss of the absorption peak at 438 nm, which was restored by the addition of 2‐oxoglutarate, thus indicating that the prosthetic group is functional in catalysis. Classical hyperbolic kinetics with substrate inhibition was seen for 2‐oxoglutarate. The Km values determined for glutamine and ferredoxin were 0.7 mM and 7 μM, respectively, and the apparent Km for 2‐oxoglutarate was estimated to be 1.7 mM. Azaserine and 6‐diazo‐5‐oxo‐l‐norleucine were potent inhibitors of the activity, while pyridoxal 5‐phosphate, known to react with Lys residues, partially inactivated the enzyme. This ferredoxin‐dependent glutamate synthase is, as far as we know, the first purified from prokaryotic organisms and resembles its counterpart from chloroplasts, suggesting that cyanobacterial glutamate synthase may have been the ancestor of ferredoxin‐glutamate synthase in plants.

Bibliography

MARQUÃS, S., FLORENCIO, F. J., & CANDAU, P. (1992). Purification and characterization of the ferredoxinâglutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301. European Journal of Biochemistry, 206(1), 69â77. Portico.

Authors 3

Silvia MARQUÉS (first)
Francisco J. FLORENCIO (additional)
Pedro CANDAU (additional)

References 42 Referenced 42

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Dates

Type	When
Created	20 years, 6 months ago (March 4, 2005, 1:34 a.m.)
Deposited	1 year, 9 months ago (Nov. 23, 2023, 2:15 a.m.)
Indexed	3 months, 2 weeks ago (May 22, 2025, 5:48 a.m.)
Issued	33 years, 4 months ago (May 1, 1992)
Published	33 years, 4 months ago (May 1, 1992)
Published Online	20 years, 6 months ago (March 3, 2005)
Published Print	33 years, 4 months ago (May 1, 1992)

Funders 0

None

BibTeX

@article{MARQU_S_1992, title={Purification and characterization of the ferredoxin‐glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301}, volume={206}, ISSN={1432-1033}, url={http://dx.doi.org/10.1111/j.1432-1033.1992.tb16902.x}, DOI={10.1111/j.1432-1033.1992.tb16902.x}, number={1}, journal={European Journal of Biochemistry}, publisher={Wiley}, author={MARQUÉS, Silvia and FLORENCIO, Francisco J. and CANDAU, Pedro}, year={1992}, month=may, pages={69–77} }

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