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A loop unique to ferredoxin-dependent glutamate synthases is not absolutely essential for ferredoxin-dependent catalytic activity

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Abstract

It had been proposed that a loop, typically containing 26 or 27 amino acids, which is only present in monomeric, ferredoxin-dependent, “plant-type” glutamate synthases and is absent from the catalytic α-subunits of both NADPH-dependent, heterodimeric glutamate synthases found in non-photosynthetic bacteria and NADH-dependent heterodimeric cyanobacterial glutamate synthases, plays a key role in productive binding of ferredoxin to the plant-type enzymes. Site-directed mutagenesis has been used to delete the entire 27 amino acid-long loop in the ferredoxin-dependent glutamate synthase from the cyanobacterium Synechocystis sp. PCC 6803. The specific activity of the resulting loopless variant of this glutamate synthase, when reduced ferredoxin serves as the electron donor, is actually higher than that of the wild-type enzyme, suggesting that this loop is not absolutely essential for efficient electron transfer from reduced ferredoxin to the enzyme. These results are consistent with the results of an in-silico study that suggests that the loop is unlikely to interact directly with ferredoxin in the energetically most favorable model of a 1:1 complex of ferredoxin with the wild-type enzyme.

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Abbreviations

CD:

Circular dichroism

Fd:

Ferredoxin

FNR:

Ferredoxin:NADP+ oxidoreductase

GOGAT:

Glutamine:oxoglutarate amidotransferase

ICP:

Inductively coupled plasma

MALDI-TOF:

Matrix-assisted laser desorption ionization, time-of-flight

MS:

Mass spectrometry

MV:

Methyl viologen

PMSF:

Phenylmethanesulfonyl fluoride

SDS-PAGE:

Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate

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Acknowledgments

The mutagenesis, protein expression and purification, kinetic measurements, substrate-binding determinations, and data analysis carried out at Texas Tech University were funded by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy, through Grant DE-FG03-99ER20346 (to D. B. K.). Sequence alignment comparisons and activity measurements on cell-free extracts of Synechocystis sp. PCC 6803 were funded by a grant (BFU2010-15708 to F. J. F.) from the Ministerio de Economia y Competitividad of Spain and European Regional Funds (FEDER). The authors would like to thank Prof. James P. Allen (Department of Chemistry and Biochemistry, Arizona State University) for carrying out the iron content determinations. The authors would also like to thank Dr. Bernard Lagoutte (CEA Saclay) for the gift of the clone used to express Synechocystis sp. PCC 6803 FNR. The authors would like to acknowledge the assistance of Ms. Mahima Kruthiventi in some of the initial mutagenesis experiments. Ms. Afia Dasgupta, Ms. Mahima Kruthiventi, and Ms. Nanditha Vaidyanathan were supported, in part, by graduate fellowships from the Texas Tech University Graduate School.

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Authors and Affiliations

  1. Center for Biotechnology and Genomics, Texas Tech University, Lubbock, TX, 79409-3132, USA

    Jatindra N. Tripathy, Afia Dasgupta, Nanditha Vaidyanathan, Masoud Zabet-Moghaddam & David B. Knaff

  2. Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409-1061, USA

    Masakazu Hirasawa, Anurag P. Srivastava & David B. Knaff

  3. Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX, 79430-6551, USA

    R. Bryan Sutton

  4. Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, 79430-6551, USA

    R. Bryan Sutton

  5. Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, 41092, Seville, Spain

    Francisco J. Florencio

Authors
  1. Jatindra N. Tripathy
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  2. Masakazu Hirasawa
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  3. R. Bryan Sutton
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  4. Afia Dasgupta
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  5. Nanditha Vaidyanathan
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  6. Masoud Zabet-Moghaddam
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  7. Francisco J. Florencio
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  8. Anurag P. Srivastava
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  9. David B. Knaff
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Corresponding author

Correspondence to David B. Knaff.

Additional information

Jatindra N. Tripathy and Masakazu Hirasawa contributed equally to this project.

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Tripathy, J.N., Hirasawa, M., Sutton, R.B. et al. A loop unique to ferredoxin-dependent glutamate synthases is not absolutely essential for ferredoxin-dependent catalytic activity. Photosynth Res 123, 129–139 (2015). https://doi.org/10.1007/s11120-014-0044-2

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