Altered Intersubunit Interactions in Crystal Structures of Catalytically Compromised Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

Substitution of Leu290 by Phe (L290F) in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from the unicellular green alga Chlamydomonas reinhardtii causes a 13% decrease in CO2/O2 specificity and reduced thermal stability. Genetic selection for restored photosynthesis at the rest...

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Published inBiochemistry (Easton) Vol. 44; no. 1; pp. 113 - 120
Main Authors Karkehabadi, Saeid, Taylor, Thomas C, Spreitzer, Robert J, Andersson, Inger
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 11.01.2005
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Summary:Substitution of Leu290 by Phe (L290F) in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from the unicellular green alga Chlamydomonas reinhardtii causes a 13% decrease in CO2/O2 specificity and reduced thermal stability. Genetic selection for restored photosynthesis at the restrictive temperature identified an Ala222 to Thr (A222T) substitution that suppresses the deleterious effects of the original mutant substitution to produce a revertant enzyme with improved thermal stability and kinetic properties virtually indistinguishable from that of the wild-type enzyme. Because the mutated residues are situated ∼19 Å away from the active site, they must affect the relative rates of carboxylation and oxygenation in an indirect way. As a means for elucidating the role of such distant interactions in Rubisco catalysis and stability, we have determined the crystal structures of the L290F mutant and L290F/A222T revertant enzymes to 2.30 and 2.05 Å resolution, respectively. Inspection of the structures reveals that the mutant residues interact via van der Waals contacts within the same large subunit (intrasubunit path, 15.2 Å Cα−Cα) and also via a path involving a neighboring small subunit (intersubunit path, 18.7 Å Cα−Cα). Structural analysis of the mutant enzymes identified regions (residues 50−72 of the small subunit and residues 161−164 and 259−264 of the large subunit) that show significant and systematically increased atomic temperature factors in the L290F mutant enzyme compared to wild type. These regions coincide with residues on the interaction paths between the L290F mutant and A222T suppressor sites and could explain the temperature-conditional phenotype of the L290F mutant strain. This suggests that alterations in subunit interactions will influence protein dynamics and, thereby, affect catalysis.
Bibliography:This work was supported by grants from the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning (FORMAS), the European Union (No. QLK3-CT-2002-01945), and the U.S. Department of Agriculture National Research Initiative (No. 2001-35318-11267).
Coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 1UWA for the L290F single mutant and 1UW9 for the L290F/A222T double mutant.
ark:/67375/TPS-BD4R0VN5-H
istex:81D4ADB663813C428C354A48407CEAD2E47635A8
ISSN:0006-2960
1520-4995
DOI:10.1021/bi047928e