Team:Bielefeld-CeBiTec/Results/CO2-fixation/CalvinCycle

Calvin cycle

The Calvin cycle as one of the important cycles for carbon dioxide fixation got our main focus. We identified the three missing enzymes for enabling the whole cycle in E.coli and did some further research about them.

Sedoheptulose 1,7-bisphosphatase (glpX)

Reaction of sedoheptulose 1,7-bisphosphatase

The SBPase is one of enzymes needed for the Calvin cycle. It catalyzes the reaction from sedoheptulose 1,7-bisphosphate to sedoheptulose 7-phosphate. The enzyme is characteristic for the part of regeneration in the Calvin-cycle. It was shown before that oveerexpression of the SBPase in tobacco results in enhanced carbon assimilation and crop yield (Rosenthal et al., 2011). SBPases are homodimeric with two identical subunits of 35kD to 38kD. The k_m-value of GlpX (Bacillus methanolicus) is 14 ± 0.5 µM (Stolzenberger et al., 2013).
It does not occur in E.coli which makes it a target to transform for enabling the whole cycle.

Phosphoribulokinase A

Reaction of phosphoribulokinase

The phosphoribulokinase A is the enzyme which catalyzes the reaction from ribulose 5-phosphate to ribulose 1,5-bisphosphate. This step needs ATP.

Ribulose 1,5-bisphosphate Carboxylase Oxygenase (RuBisCO)

Reaction of phosphoribulokinase

The Ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) is the most abundant enzyme in the world. Because of its key role in carbon fixation metabolism, it is found in nearly all autotrophic organisms like plants, but also in cyanobacteria and fotosynthetic bacteria in high concentrations (Andersson, 2008). RuBisCo catalyses the fixation of atmospheric carbon dioxide by generating two tricarbohydrates out of one pentacarbohydrate. So you could say it is responsible for conversion of carbon dioxide in biomass or with other words for incorporation of inorganic carbon dioxide to form organic molecules. To give some facts, more than 10¹¹ tons of atmospheric carbon dioxide is fixed per year baesd on RuBisCo activity (Field et al., 1998).
RuBisCo catalyses the rate limiting step in the Calvin cycle. The Calvin cycle is the light independent reaction of photosynthesis. In this cycle, carbon dioxide is fixed to build up energy-rich substrates. RuBisCo catalyses the fixation of one molecule carbon dioxide to ribulose-1,5-bisphosphate (RuBP). The product is instabile and decays directly in two molecules 3-D-phosphoglycerate (3-PGA)(Andersson, 2008, Parikh et al. 2006). 3-PGA is further converted in the Calvin cycle to glycerinaldehyde-3-phosphate and metabolized by the cells. Furthermore 3-D-phosphoglycerate is an essential intermediate in the central metabolism, as it plays a central role in glycolysis and gluconeogenesis.
Beside the carbon fixation reaction of the RuBisCo, the enzyme catalyses numerous side reactions. An alternative substrate to carbon dioxide is atmospheric oxygen. When the oxygenation of RuBP ist catalyzed instead of carboxylation, the product is 2-phosphoglycolate. This product can not be used by the metabolism of the cells and the fixed carbon has to be regenerated by the metabolic pathway photorespiration, a high-energy consuming pathway. This metabolic stress for the cells reduces the effizienz of carbon dioxide fixation about 20 - 50 % (Mann, 1999, Andersson, 2008).
The catalyzed carboxylation/ oxygenation of RuBP is a multiple step reaction. In detail, the firt step is activation of the RuBisCo by carbamylation of the amino group from a Lysin in the active centre. The activated RuBisCo is then stabilized by magnesium ions, a cofactor for activity. In the carboxylation/ oxygenation of RuBP the first step is enolisation of the substrate by H₂O. The enediolate reacts then with either carbon dioxide or oxygen. If carbon dioxide is bound by the enediolate, in the next water is split up from the molecule and the instabile intermediate decomposes in two molecules 3-PGA. If oxygen is bound by the enediolate, two protons are split of and 2-phosphoglycolate is build up. (Andersson, 2008)
The competing reaction between CO₂ and O₂ limits the efficiency of the RuBisCo. The oxygenation side reaction of the RuBisCo is one reason for the inefficiency of this enzyme. The higher affinity of RuBisCo to carbon dioxide, nearly by a factor 100 higher than to oxygen (in higher plants), still does not have a positive effect for the efficiency, because of the concentration from both gases in the atmosphere. Oxygen has a percentage of 20 % (v/v) whereas carbon dioxide accounts for only 0,04 % (v/v).

The Ribulose 1,5-bisphosphate carboxylase oxygenase is the most abundant enzyme of the world because it occurs in every plant in a high concentration. The reaction of this enzyme is essential for the functionality of the Calvin cycle because it uses the atmospheric carbon dioxide to generate two tricarbohydrates out of one pentacarbohydrate. The problem of the RuBisCO is that is also accepts oxygen with a higher percentage as cofactor. The following reaction results in one dicarbohydrate and one tricarbohydrate.
The RuBisCO consists of two subunits, a small and a large subunit. In higher plants the RuBisCO is formed out of four large and four small subunits. In smaller organisms the RuBisCO is only formed out of two proteins each.
We aim to use the RuBisCO from two different organisms which are mentioned below.