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Degradation of potent Rubisco inhibitor by selective sugar phosphatase

Nature Plants volume 1, Article number: 14002 (2015) | Download Citation

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  • An Erratum to this article was published on 14 January 2015

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the conversion of atmospheric carbon dioxide into organic compounds in photosynthetic organisms. Alongside carboxylating the five-carbon sugar ribulose-1,5-bisphosphate (RuBP)1,​2,​3, Rubisco produces a small amount of xylulose-1,5-bisphosphate (XuBP), a potent inhibitor of Rubisco4. The AAA+ protein Rubisco activase removes XuBP from the active site of Rubisco in an ATP-dependent process5,6. However, free XuBP rapidly rebinds to Rubisco, perpetuating its inhibitory effect. Here, we combine biochemical and structural analyses to show that the CbbY protein of the photosynthetic bacterium Rhodobacter sphaeroides and Arabidopsis thaliana is a highly selective XuBP phosphatase. We also show that CbbY converts XuBP to the non-inhibitory compound xylulose-5-phosphate, which is recycled back to RuBP. We solve the crystal structures of CbbY from R. sphaeroides and A. thaliana, and through mutational analysis show that the cap domain of the protein confers the selectivity for XuBP over RuBP. Finally, in vitro experiments with CbbY from R. sphaeroides reveal that CbbY cooperates with Rubisco activase to prevent a detrimental build-up of XuBP at the Rubisco active site. We suggest that CbbY, which is conserved in algae and plants, is an important component of the cellular machinery that has evolved to deal with the shortcomings of the ancient enzyme Rubisco.

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References

  1. 1.

    & Rubisco: structure, regulatory interactions, and possibilities for a better enzyme. Ann. Rev. Plant Biol. 53, 449–475 (2002).

  2. 2.

    & Structure and function of Rubisco. Plant Physiol. Biochem. 46, 275–291 (2008).

  3. 3.

    et al. Rubisco activity and regulation as targets for crop improvement. J. Exp. Bot. 64, 717–730 (2013).

  4. 4.

    Catalytic by-product formation and ligand binding by ribulose bisphosphate carboxylases from different phylogenies. Biochem. J. 399, 525–534 (2006).

  5. 5.

    Rubisco activase – Rubisco's catalytic chaperone. Photosynth. Res. 75, 11–27 (2003).

  6. 6.

    , , , & Rubisco regulation: a role for inhibitors. J. Exp. Bot. 59, 1569–1580 (2008).

  7. 7.

    & Analysis of the cbbXYZ operon in Rhodobacter sphaeroides. J. Bacteriol. 179, 663–669 (1997).

  8. 8.

    & Computer analysis of bacterial haloacid dehalogenases defines a large superfamily of hydrolases with diverse specificity. Application of an iterative approach to database search. J. Mol. Biol. 244, 125–132 (1994).

  9. 9.

    et al. AT_CHLORO, a comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins. Mol. Cell. Proteomics 9, 1063–1084 (2010).

  10. 10.

    et al. Sorting signals, N-terminal modifications and abundance of the chloroplast proteome. PLoS ONE 3, e1994 (2008).

  11. 11.

    & Photosynthesis and ribulose 1,5-bisphosphate levels in intact chloroplasts. Plant Physiol. 64, 880–883 (1979).

  12. 12.

    et al. 2-carboxy-d-arabinitol 1-phosphate (CA1P) phosphatase: evidence for a wider role in plant Rubisco regulation. Biochem. J. 442, 733–742 (2012).

  13. 13.

    , , & The pentacovalent phosphorus intermediate of a phosphoryl transfer reaction. Science 299, 2067–2071 (2003).

  14. 14.

    et al. Analysis of the structural determinants underlying discrimination between substrate and solvent in β-phosphoglucomutase catalysis. Biochem. 48, 1984–1995 (2009).

  15. 15.

    et al. Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF-3 rather than by phosphoranes. Proc. Natl Acad. Sci. USA 107, 4555–4560 (2010).

  16. 16.

    , , & Caught in the act: the structure of phosphorylated beta-phosphoglucomutase from Lactococcus lactis. Biochemistry 41, 8351–8359 (2002).

  17. 17.

    & Reversible inactivation and characterization of purified inactivated form I ribulose 1,5-bisphosphate carboxylase/oxygenase of Rhodobacter sphaeroides. J. Bacteriol. 174, 3593–3600 (1992).

  18. 18.

    , & The activation of ribulose-1,5-bisphosphate carboxylase by carbon dioxide and magnesium ions. Equilibria, kinetics, a suggested mechanism, and physiological implications. Biochemistry 15, 529–536 (1976).

  19. 19.

    & Fallover of ribulose 1,5-bisphosphate carboxylase/oxygenase activity: decarbamylation of catalytic sites depends on pH. Plant Physiol. 97, 1354–1358 (1991).

  20. 20.

    & Xylulose 1,5-bisphosphate synthesized by ribulose 1,5-bisphosphate carboxylase/oxygenase during catalysis binds to decarbamylated enzyme. Plant Physiol. 97, 1348–1353 (1991).

  21. 21.

    , , & The Calvin cycle enzyme pentose-5-phosphate 3-epimerase is encoded within the cfx operons of the chemoautotroph Alcaligenes eutrophus. J. Bacteriol. 174, 7337–7344 (1992).

  22. 22.

    , , & The cbb operons of the facultative chemoautotroph Alcaligenes eutrophus encode phosphoglycolate phosphatase. J. Bacteriol. 175, 7329–7340 (1993).

  23. 23.

    , , & Role of metal ions in the reaction catalysed by l-ribulose-5-phosphate 4-epimerase. Biochemistry 39, 4821–4830 (2000).

  24. 24.

    et al. Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase. Nature 479, 194–199 (2011).

  25. 25.

    , , , & An efficient system for high-level expression and easy purification of authentic recombinant proteins. Protein Sci. 13, 1331–1339 (2004).

  26. 26.

    , & A malachite green colorimetric assay for protein phosphatase activity. Anal. Biochem. 192, 112–116 (1991).

  27. 27.

    & Adenosine triphosphate hydrolysis by purified Rubisco activase. Arch. Biochem. Biophys. 268, 93–99 (1989).

  28. 28.

    et al. MgF3 and α-galactose 1-phosphate in the active site of β-phosphoglucomutase form a transition state analogue of phosphoryl transfer. J. Am. Chem. Soc. 131, 16334–16335 (2009).

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Acknowledgements

We thank J. Soll (LMU Munich, Germany) for A. thaliana cDNA, J. Andralojc and M. Parry (Rothamsted, England) for the initial XuBP, and the JSBG group at ESRF Grenoble, as well as staff at SLS-X06DA and X10SA Villigen, MPIB Crystallization Facility and MPIB Core Facility for their excellent support.

Author information

Author notes

    • Andreas Bracher
    •  & Anurag Sharma

    These authors contributed equally to this work

    • Amanda Starling-Windhof

    Present address: Chemical Abstracts Service, 2540 Olentangy River Road, Columbus, Ohio 43202, USA

Affiliations

  1. Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

    • Andreas Bracher
    • , Anurag Sharma
    • , Amanda Starling-Windhof
    • , F. Ulrich Hartl
    •  & Manajit Hayer-Hartl

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Contributions

A.B. solved the crystal structures and planned and supervised the experiments. A.S. designed and performed the experiments. The initial crystal screening and preliminary phosphatase analysis was performed by A-S.W. The project was initiated by M.H-H. and F.U.H. and M.H-H. planned and supervised the project. All authors analysed the data and the manuscript was written by A.B., F.U.H. and M.H-H.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Andreas Bracher or Manajit Hayer-Hartl.

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DOI

https://doi.org/10.1038/nplants.2014.2

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