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NanoESI mass spectrometry of Rubisco and Rubisco activase structures and their interactions with nucleotides and sugar phosphates

Journal Article


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Abstract


  • Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is the protein that is responsible for the fixation of carbon dioxide in photosynthesis. Inhibitory sugar phosphate molecules, which can include its substrate ribulose-1,5- bisphosphate (RuBP), can bind to Rubisco catalytic sites and inhibit catalysis. These are removed by interaction with Rubisco activase (RA) via an ATP hydrolytic reaction. Here we show the first nanoESI mass spectra of the hexadecameric Rubisco and of RA from a higher plant (tobacco). The spectra of recombinant, purified RA revealed polydispersity in its oligomeric forms (up to hexamer) and that ADP was bound. ADP was removed by dialysis against a high ionic strength solution and nucleotide binding experiments showed that ADP bound more tightly to RA than AMP-PNP (a non-hydrolysable ATP analog). There was evidence that there may be two nucleotide binding sites per RA monomer. The oligomerization capacity of mutant and wild-type tobacco RA up to hexamers is analogous to the subunit stoichiometry for other AAA+ enzymes. This suggests assembly of RA into hexamers is likely the most active conformation for removing inhibitory sugar phosphate molecules from Rubisco to enable its catalytic competency. Stoichiometric binding of RuBP or carboxyarabinitol bisphosphate (CABP) to each of the eight catalytic sites of Rubisco was observed. © 2011 American Society for Mass Spectrometry.

Authors


  •   Blayney, Michelle (external author)
  •   Whitney, Spencer M. (external author)
  •   Beck, Jennifer L.

Publication Date


  • 2011

Citation


  • Blayney, M. J., Whitney, S. M. & Beck, J. L. (2011). NanoESI mass spectrometry of Rubisco and Rubisco activase structures and their interactions with nucleotides and sugar phosphates. Journal of the American Society for Mass Spectrometry, 22 (9), 1588-1601.

Scopus Eid


  • 2-s2.0-80052686543

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=6731&context=scipapers

Ro Metadata Url


  • http://ro.uow.edu.au/scipapers/3389

Has Global Citation Frequency


Number Of Pages


  • 13

Start Page


  • 1588

End Page


  • 1601

Volume


  • 22

Issue


  • 9

Abstract


  • Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is the protein that is responsible for the fixation of carbon dioxide in photosynthesis. Inhibitory sugar phosphate molecules, which can include its substrate ribulose-1,5- bisphosphate (RuBP), can bind to Rubisco catalytic sites and inhibit catalysis. These are removed by interaction with Rubisco activase (RA) via an ATP hydrolytic reaction. Here we show the first nanoESI mass spectra of the hexadecameric Rubisco and of RA from a higher plant (tobacco). The spectra of recombinant, purified RA revealed polydispersity in its oligomeric forms (up to hexamer) and that ADP was bound. ADP was removed by dialysis against a high ionic strength solution and nucleotide binding experiments showed that ADP bound more tightly to RA than AMP-PNP (a non-hydrolysable ATP analog). There was evidence that there may be two nucleotide binding sites per RA monomer. The oligomerization capacity of mutant and wild-type tobacco RA up to hexamers is analogous to the subunit stoichiometry for other AAA+ enzymes. This suggests assembly of RA into hexamers is likely the most active conformation for removing inhibitory sugar phosphate molecules from Rubisco to enable its catalytic competency. Stoichiometric binding of RuBP or carboxyarabinitol bisphosphate (CABP) to each of the eight catalytic sites of Rubisco was observed. © 2011 American Society for Mass Spectrometry.

Authors


  •   Blayney, Michelle (external author)
  •   Whitney, Spencer M. (external author)
  •   Beck, Jennifer L.

Publication Date


  • 2011

Citation


  • Blayney, M. J., Whitney, S. M. & Beck, J. L. (2011). NanoESI mass spectrometry of Rubisco and Rubisco activase structures and their interactions with nucleotides and sugar phosphates. Journal of the American Society for Mass Spectrometry, 22 (9), 1588-1601.

Scopus Eid


  • 2-s2.0-80052686543

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=6731&context=scipapers

Ro Metadata Url


  • http://ro.uow.edu.au/scipapers/3389

Has Global Citation Frequency


Number Of Pages


  • 13

Start Page


  • 1588

End Page


  • 1601

Volume


  • 22

Issue


  • 9