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Substitutions at the opening of the Rubisco central solvent channel affect holoenzyme stability and CO2/O2 specificity but not activation by Rubisco activase

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Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the initial step of carbon metabolism in photosynthesis. The holoenzyme comprises eight large subunits, arranged as a tetramer of dimers around a central solvent channel that defines a fourfold axis of symmetry, and eight small subunits, arranged as two tetramers at the poles of the axis. The phylogenetically divergent small-subunit loops between β-strands A and B form the entrance to the solvent channel. In the green alga Chlamydomonas reinhardtii, Ile-58 from each of the four small-subunit βA–βB loops defines the minimal diameter of the channel opening. To understand the role of the central solvent channel in Rubisco function, directed mutagenesis and transformation of Chlamydomonas were employed to replace Ile-58 with Ala, Lys, Glu, Trp, or three Trp residues (I58W3) to close the entrance to the channel. The I58E, I58K, and I58W substitutions caused only small decreases in photosynthetic growth at 25 and 35 °C, whereas I58W3 had a substantial effect at both temperatures. The mutant enzymes had decreased carboxylation rates, but the I58W3 enzyme had decreases in both carboxylation and CO2/O2 specificity. The I58E, I58W, and I58W3 enzymes were inactivated at lower temperatures than wild-type Rubisco, and were degraded at slower rates under oxidative stress. However, these mutant enzymes were activated by Rubisco activase at normal rates, indicating that the structural transition required for carboxylation is not affected by altering the solvent channel opening. Structural dynamics alone may not be responsible for these distant effects on the Rubisco active site.

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Acknowledgments

This study was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences, of the United States Department of Energy through Photosynthetic Systems Grants DE-FG02-00ER15044 to R.J.S. and DE-AI02-97ER20268 to M.E.S. We acknowledge the technical assistance of Dr. Csengele Barta (USDA-ARS, Maricopa, AZ) for the Rubisco activation assays.

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  1. Todor Genkov

    Present address: Center for Biocatalysis and Bioprocessing, University of Iowa, Coralville, IA, 52241, USA

Authors and Affiliations

  1. Instituto Superior de Agronomia (ISA), Technical University of Lisbon, 1399, Lisbon, Portugal

    M. Gloria Esquivel & Ana S. Nogueira

  2. Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588, USA

    Todor Genkov & Robert J. Spreitzer

  3. Arid-Land Agricultural Research Center, Agricultural Research Service, US Department of Agriculture, Maricopa, AZ, 85138, USA

    Michael E. Salvucci

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  1. M. Gloria Esquivel
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Correspondence to M. Gloria Esquivel.

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Esquivel, M.G., Genkov, T., Nogueira, A.S. et al. Substitutions at the opening of the Rubisco central solvent channel affect holoenzyme stability and CO2/O2 specificity but not activation by Rubisco activase. Photosynth Res 118, 209–218 (2013). https://doi.org/10.1007/s11120-013-9916-0

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