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Team:Bielefeld-CeBiTec/Results/CO2-fixation/CalvinCycle
From 2014.igem.org
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 km-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 one molecule 3-PGA and one molecule 2-phosphoglycolate. 2-phosphoglycolate has only limited use for the metabolism of the cells and the fixed carbon has to be regenerated by a metabolic pathway called photorespiration, a high-energy consuming pathway. In photorespiration, two molecules 2-phosphoglycolate are split up in one molecule 3-PGA and one molecule carbon dioxide. 3-PGA can enter the Calvin cycle, whereas CO2 is unusable for the cells. Because of the oxygenation side reaction the effiziency of carbon dioxide fixation by RuBisCo is reduced about 20 - 50 % (Andersson, 2008) (Mann, 1999).
The catalyzed carboxylation/ oxygenation of RuBP is a multiple step reaction. In detail, the first 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 and enol-RuBP is build up. The enediolate reacts then in an irreversible, partial reaction with either carbon dioxide or oxygen. This reaction determines the specifity and the rate of carbon dioxide fixation as well as the effizenz. If carbon dioxide is bound by the enediolate, the instabile intermediate is protonated and hydrated to build up two molecules 3-PGA. If oxygen is bound by the enediolate, the intermediate decomposes directyl in 3-phosphoglycerate and 2-phosphoglycolate. (Andersson, 2008), (Spreitzer, Salvucci 2002)
The competing reaction between CO2 and O2 and the resulting oxygenation side reaction limits the efficiency of RuBisCo. The effiziency is often quantified by a specifity factor. This is the ration of the catalytic efficiency of carboxylation to oxygenation, described by the maximal velocities of carboxylation and oxygenation, and the Michaelis-Menten constants for carbon dioxide and oxygen. (Andersson, 2008) (Jordan, Ogren 1981) (Spreitzer, Salvucci 2002) The specifity factors of RuBisCo enzymes differ significantly depending on the host organism of the RuBisCo. Bacteria have low specifity factors in comparison to higher plants oder algae. As there exist a inverse correlation between turnover rate (for carboxylation) and specifity factor, Bacteria have low specifity factors, but high turnover rates. Higher organism are characterized by high specifity factors and low turnover rates. (Andersson, 2008) (Jordan, Ogren 1981)
Carboxysomes, bacterial microcompartiments, are one mechanism for carbon dioxide concentration. By expression of carboxysomes the carbon fixation efficiency is increased, using the high turnover rates. (Bonacci et al., 2011)
RuBisCo is a multiprotein enzyme, containing two types of subunits, the large (L) subunit (50-55 kDa) and the small (S) subunit (12-18 kDa). The most common form of RuBisCo (form I or form IA) consist of eight large subunits, which are forming dimers, and eight small subunits. Together they form a hexadimeric structure. Form I occurs in most autotrophic bacteria, algae and higher plants. The large subunit is the catalytic one, and the small subunit is not essential for catalysis. The octamer of large subunit still remains carboxylation activity.
RuBisCo form II or form IB is found in some chemoautotrophic bacteria and in dinoflagellates. This form is characterized by the abscence of the small subunits. (Andersson, 2008) (Spreitzer, Salvucci 2002)
