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Structural analysis of altered large-subunit loop-6/carboxy-terminus interactions that influence catalytic efficiency and CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase.
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2007 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 39, 11080-11089 p.Article in journal (Refereed) Published
Abstract [en]

The loop between alpha-helix 6 and beta-strand 6 in the alpha/beta-barrel of ribulose-1,5-bisphosphate carboxylase/oxygenase plays a key role in discriminating between CO2 and O2. Genetic screening in Chlamydomonas reinhardtii previously identified a loop-6 V331A substitution that decreases carboxylation and CO2/O2 specificity. Revertant selection identified T342I and G344S substitutions that restore photosynthetic growth by increasing carboxylation and specificity of the V331A enzyme. In numerous X-ray crystal structures, loop 6 is closed or open depending on the activation state of the enzyme and the presence or absence of ligands. The carboxy terminus folds over loop 6 in the closed state. To study the molecular basis for catalysis, directed mutagenesis and chloroplast transformation were used to create T342I and G344S substitutions alone. X-ray crystal structures were then solved for the V331A, V331A/T342I, T342I, and V331A/G344S enzymes, as well as for a D473E enzyme created to assess the role of the carboxy terminus in loop-6 closure. V331A disturbs a hydrophobic pocket, abolishing several van der Waals interactions. These changes are complemented by T342I and G344S, both of which alone cause decreases in CO2/O2 specificity. In the V331A/T342I revertant enzyme, Arg339 main-chain atoms are displaced. In V331A/G344S, alpha-helix 6 is shifted. D473E causes disorder of the carboxy terminus, but loop 6 remains closed. Interactions between a transition-state analogue and several residues are altered in the mutant enzymes. However, active-site Lys334 at the apex of loop 6 has a normal conformation. A variety of subtle interactions must be responsible for catalytic efficiency and CO2/O2 specificity.

Place, publisher, year, edition, pages
2007. Vol. 46, no 39, 11080-11089 p.
National Category
Natural Sciences
Research subject
Biology with specialization in Structural Biology
Identifiers
URN: urn:nbn:se:uu:diva-292928DOI: 10.1021/bi701063fPubMedID: 17824672OAI: oai:DiVA.org:uu-292928DiVA: diva2:926980
Available from: 2016-05-10 Created: 2016-05-10 Last updated: 2016-05-10

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