Conner Tippetts

Title: Structural mechanism of RuBisCO activation by carbamylation of the active site

lysine

Introduction: RuBisCO (Ribulose-1, 5-bisphosphate carboxylase/oxygenase) is the most
abundant protein on earth. This abundance is made up by the fact that it is a relatively inefficient
enzyme. It has been recently hypothesized that RuBisCO has evolved as an inefficient enzyme
because it is compensated by its abundance. Rubisco is the leading contributor in converting CO
2

into organic compounds via the Calvin cycle. RuBisCO activation has not been studied
significantly.

Reasons for research: The aim of the study was to define the mechanism of RuBisCO
activation and principles of ligand discrimination of CO
2
and O
2
(two molecules that bind to one
of the enzymes active sites).
As a result, this research provides a basis on which efficiency improvements and general
catalytic activity of the enzyme can follow. Knowledge of these elements can also be used in
engineering crop enhancement and possibly contributing to a global warming remedy.

Methods and Materials: The enzyme was initially isolated from primarily G. sulphuraria and
treated with 20mM of MgSO
4
. This was done at both room temperature and ~40 C. Data was
collected using Microfocus 007 and Raxis IV++ imaging plate detector. The structures were then
refined using Shelx93 and Refmac. These tools further enhance the image of the gaseous ligands
(O
2
and CO
2
). Final refinement was completed using excellent stereochemistry.
The ligands were identified using elimination of residual difference identities, using
comparable levels of B-factors of surrounding atoms, and understanding proper hydrogen
bonding patterns. All of this contributed to creating the most accurate model of the enzyme, as
well as interactions of ligands by discrimination and achieving final results.

Results: The structure of the catalytic domain or active site of RuBisCO was found to be an /
(TIM) barrel. This was demonstrated by the lack of substrate for the enzyme to bind to, thus
giving a better view of the catalytic domain. The active site cavity was also lined with highly
mobile loops for binding to gaseous ligands/substrates (O
2
and CO
2
).
The presence of nitrosylated (similar to being phosphorylated) Cys residues was
unexpected seeing as this modification has not been reported in G. sulphuraria RuBisCO. It is
usually found in higher eukaryotes, but this evidence suggests otherwise.

Discussion: In the enzyme RuBisCO, ligands are trapped because the enzyme is inactivated by
nitrosylation. This effect of nitrosylation controlling the effects of RuBisCO activities is one that
has not received sufficient attention.
Dimeric forms of RuBisCO are relatively easy material to genetically manipulate. This is
probably the most practical way of reengineering the enzyme for future improvements and
applications.

Conclusion: Knowledge of the structure of the active site and method of activation of RuBisCO
are necessary to further improve the function of the enzyme. Crop efficiency or enhancement
will come with a RuBisCO enzyme that can differentiate between gaseous ligands with more
efficiency and increasing its catalytic reactions. Aiding in the remedy for global warming, as
mentioned earlier, can be achieved through improving the active sites function of activation to
be more productive. Through a reductionist mindset, one can see the possibilities arising from
the identification of the active site.

Limitations of the study: RuBisCO was only isolated from a selective body of sources,
primarily the red algae Galdieria sulphuraria, thus limiting the results to the selected class of
plants.

Bibliography: http://www.pnas.org/content/109/46/18785.full.pdf