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

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   <h6>Calvin cycle</h6>
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     <p>The Calvin cycle, shown in Figure 1, is quantitatively one of the most important pathways for carbon dioxide fixation in photoautotrophic organisms (<a href="#huegler2011">Hügler and Sievert, 2011</a>). High seperation from other metabolic cycles, like central carbon mechanism, allows an effective regulation by the cells. Three enzymes are characteristic for this cycle. The Ribulose 1,5-bisphosphate Carboxylase Oxygenase (RuBisCO), Phosphoribulokinase A (prkA) and Sedoheptulose 1,7-bisphosphatase, described all in detail below. The starting substance is Ribulose-1,5-bisphosphate, which is converted in enzymatic reactions in glyceraldehyde-3-phosphate. The regeneration part consists of the interconversion of triose phosphates to Ribulose-1,5-bisphosphate, containing reactions of the prkA and SBPase. As a summary, in the Calvin cycle six molecules of NADPH and nine molecules of ATP are required for building up one molecule of glyceraldehyde-3-phosphate.
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     <p>The Calvin cycle, shown in Figure 1, is quantitatively one of the most important pathways for carbon dioxide fixation in photoautotrophic organisms (<a href="#huegler2011">Hügler and Sievert, 2011</a>). It is separated in three parts: Carboxylation, reduction and regeneration. High separation from other metabolic pathways, like central carbon mechanism, allows an effective regulation by the cells. Three enzymes are characteristic for this cycle. The Ribulose 1,5-bisphosphate Carboxylase Oxygenase (RuBisCO), Phosphoribulokinase A (PrkA) and Sedoheptulose 1,7-bisphosphatase (SBPase) are, described in detail below. The first substance in the Calvin cycle is Ribulose-1,5-bisphosphate, which is converted in multiple enzymatic reaction steps to glyceraldehyde-3-phosphate. The regeneration part consists of the interconversion of triose phosphates to Ribulose-1,5-bisphosphate, containing reactions of the prkA and SBPase. As a summary, in the Calvin cycle six molecules of NADPH and nine molecules of ATP are required for building up one molecule of glyceraldehyde-3-phosphate.
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<br> Usually it uses the energy and reduction equivalents which are produced in the light reaction of the photosynthesis. Nevertheless, this molecules could also be supplied by another source. The aim of our project is electroautotrophic growth of <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>E. coli</i></a>. Electric power replaces light as energy source of our Calvin cycle. Most of the enzymes needed in this pathway are already present in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E. coli" target="_blank"><i>E. coli</i></a>. This is due to the fact that this cycle could run in both directions. <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E. coli" target="_blank"><i>E. coli</i></a> uses some parts of this pathway in another context.
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<br> Usually, in phototrophic organisms, the Calvin cycle uses the energy and reduction equivalents produced in the light reaction of the photosynthesis. Nevertheless, this molecules could also be supplied by another source. The aim of our project is electroautotrophic growth of <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>E. coli</i></a>. Electric power replaces light as energy source in our Calvin cycle. Most of the enzymes needed in this pathway are already present in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E. coli" target="_blank"><i>E. coli</i></a>. But <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E. coli" target="_blank"><i>E. coli</i></a> uses some parts of this pathway in another context.
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All in all we identified three reaction steps for which enzymes are missing. Sedoheptulose-1,7-bisphosphatase, phosphoribulokinase and RubisCO needs to be heterologous expressed. We will focus on this enzymes in the following parts.
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All in all we identified three reaction steps for which enzymes are missing (Figure 1). The enzymes Sedoheptulose-1,7-bisphosphatase, phosphoribulokinase and RubisCO need to be heterologous expressed. We will focus on these enzymes in the following parts.
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<h6>Sedoheptulose 1,7-bisphosphatase</h6>
<h6>Sedoheptulose 1,7-bisphosphatase</h6>
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     <p>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 (<a href="#rosenthal2011">Rosenthal et al., 2011</a>). SBPases are homodimeric with two identical subunits of 35kD to 38kD. The <i>K<sub>M</sub></i>-value of GlpX (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#B.methanolicus" target="_blank"><i>Bacillus methanolicus</i></a>) is 14 &plusmn; 0.5 µM (<a href="#stolzenberger2013">Stolzenberger et al., 2013</a>).<br> It does not occur in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>E. coli</i></a> which makes it a target to transform for enabling the whole cycle.</p>
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     <p>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 sedoheptulose 7-phosphate regeneration in the Calvin-cycle. It was shown before that oveerexpression of the SBPase in tobacco results in enhanced carbon assimilation and crop yield (<a href="#rosenthal2011">Rosenthal et al., 2011</a>). SBPases are homodimers with two identical subunits of 35kD to 38kD. The <i>K<sub>M</sub></i>-value of the SBPase homologue GlpX of <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#B.methanolicus" target="_blank"><i>Bacillus methanolicus</i></a> is 14 &plusmn; 0.5 µM (<a href="#stolzenberger2013">Stolzenberger et al., 2013</a>).<br>
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<i>E. coli</i> does not have a SBPase homologue which is needed <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>E. coli</i></a> for enabling the whole cycle.</p>
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Revision as of 19:05, 17 October 2014


Module II - Carbon Dioxide (CO2) Fixation

Calvin cycle

The Calvin cycle, shown in Figure 1, is quantitatively one of the most important pathways for carbon dioxide fixation in photoautotrophic organisms (Hügler and Sievert, 2011). It is separated in three parts: Carboxylation, reduction and regeneration. High separation from other metabolic pathways, like central carbon mechanism, allows an effective regulation by the cells. Three enzymes are characteristic for this cycle. The Ribulose 1,5-bisphosphate Carboxylase Oxygenase (RuBisCO), Phosphoribulokinase A (PrkA) and Sedoheptulose 1,7-bisphosphatase (SBPase) are, described in detail below. The first substance in the Calvin cycle is Ribulose-1,5-bisphosphate, which is converted in multiple enzymatic reaction steps to glyceraldehyde-3-phosphate. The regeneration part consists of the interconversion of triose phosphates to Ribulose-1,5-bisphosphate, containing reactions of the prkA and SBPase. As a summary, in the Calvin cycle six molecules of NADPH and nine molecules of ATP are required for building up one molecule of glyceraldehyde-3-phosphate.
Usually, in phototrophic organisms, the Calvin cycle uses the energy and reduction equivalents produced in the light reaction of the photosynthesis. Nevertheless, this molecules could also be supplied by another source. The aim of our project is electroautotrophic growth of E. coli. Electric power replaces light as energy source in our Calvin cycle. Most of the enzymes needed in this pathway are already present in E. coli. But E. coli uses some parts of this pathway in another context. All in all we identified three reaction steps for which enzymes are missing (Figure 1). The enzymes Sedoheptulose-1,7-bisphosphatase, phosphoribulokinase and RubisCO need to be heterologous expressed. We will focus on these enzymes in the following parts.


Figure 1: Reactions of the Calvin cycle
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 sedoheptulose 7-phosphate 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 homodimers with two identical subunits of 35kD to 38kD. The KM-value of the SBPase homologue GlpX of Bacillus methanolicus is 14 ± 0.5 µM (Stolzenberger et al., 2013).
E. coli does not have a SBPase homologue which is needed E. coli for enabling the whole cycle.


Figure 2: Reaction of sedoheptulose 1,7-bisphosphatase
Phosphoribulokinase

The phosphoribulokinase is the enzyme which catalyzes the irreversible reaction from ribulose 5-phosphate to ribulose 1,5-bisphosphate. This step is ATP dependent. The phosphoribulokinase is an enzyme unique to the Calvin cycle (Rumpho et al., 2009). All photosynthetic organisms depend on the prk to sustain their cyclic activity. Several information like regulation and protein activity are well characterized for plants and also for cyanobacteria (Wadano et al., 1995). In many organisms the prk is activated in the presence of light which is thioredoxin mediated (Reduction of intramolecular disulfides). DTT may stimulate the enzyme activity in certain cases (Hariharan et al., 1998).


Figure 3: Reaction of phosphoribulokinase
Ribulose 1,5-bisphosphate Carboxylase Oxygenase (RuBisCO)

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. This reaction is part of the Calvin Cycle. It can be stated that the RuBisCO 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 numbers, more than 1011 tons of atmospheric carbon dioxide are fixated 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), a pentacarbohydrate. The product is unstable and decays directly into two molecules of 3-D-phosphoglycerate (3-PGA)(Andersson, 2008; Parikh et al. 2006). The reaction is shown in Figure 4. 3-PGA is further converted in the Calvin cycle to glycerinaldehyde-3-phosphate. This is an essential intermediate in the central metabolism, as it plays a central role in glycolysis and gluconeogenesis.




Figure 4: Catalyzed reaction by the RuBisCO. Ribulose-1,5-bisphosphate and carbon dioxide are converted to two molecules of 3-phosphoglycerate.

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 is catalyzed instead of the carboxylation, the product is one molecule of 3-PGA and one molecule of 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 of 2-phosphoglycolate are split up into one molecule of 3-PGA and one molecule of carbon dioxide. 3-PGA can enter the Calvin cycle, whereas CO2 is an energy with a low energy content. Because of the oxygenation side reaction the efficiency of the carbon dioxide fixation rate of RuBisCO is reduced about 20 - 50 % (Andersson, 2008; Mann, 1999).

The carboxylation/ oxygenation of RuBP catalyzed by RuBisCO is a multiple step reaction. In detail, the first step is activation of the RuBisCO by carbamylation of the amino group of a Lysin in the active centre. The activated RuBisCO is then stabilized by magnesium ions, a cofactor for enzyme activity. In the carboxylation/ oxygenation of Ribulose-1,5-bisphosphate the first step is enolisation of the substrate and enol-RuBP is build up. The enediolate reacts then in an irreversible reaction with either carbon dioxide or oxygen. This reaction determines the specificity and the rate of carbon dioxide fixation as well as the efficiency. If carbon dioxide is bound to the enediolate, the unstable intermediate is protonated and hydrated to build up two molecules of 3-PGA. If oxygen is bound to the enediolate, the intermediate decomposes directly 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 efficiency is often quantified by a specificity factor. This is the ratio 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 specificity factors of various RuBisCO enzymes differ significantly depending on the host organism of the RuBisCO. Bacteria have low specificity factors in comparison to higher plants or algae. As there exist an inverse correlation between turnover rate (for carboxylation) and specificity factor, Bacteria have low specificity factors, but high turnover rates. Higher organism are characterized by high specifity factors and low turnover rates. (Andersson, 2008; Jordan, Ogren 1981)

Carboxysomes, one type of bacterial microcompartiments, are one strategy developt by evolution for carbon dioxide concentration. By expression of carboxysomes the carbon fixation efficiency in Halothiobacillus neapolitanus is increased, using the high turnover rates. (Bonacci et al., 2011)

RuBisCO is a multiprotein enzyme, which consists of 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 form 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)

Phosphofructokinase / Fructose-1,6-bisphosphatase

One step in the regeneration of ribulose-1,5-bisphosphate is catalyzed by the fructose-1,6-bisphosphatase. This reaction is also part of the glucogenesis. The FBPase removes one phosphate from the fructose-1,6-bisphosphate. Of course the direction of glucogenesis can be reverted and this process is then called glycolysis. The phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate is catalyzed by the phosphofructokinase (PFK). This reaction consumes ATP. If both enzymes were active at the same time there will be a futile cycle. The PFK uses ATP for the phosphorylation reaction and the FBPase removes the phosphate. It is not just a useless reaction, but it also consumes a lot of ATP. Therefore the activity of both enzymes is strictly controlled. The underlying mechanisms are well understood in plants. Redox states of the cells are important regulators (Michelet et al., 2013). This type of regulation allows switching between reductive pentose phosphate pathway in the light and oxidative pentose phosphate pathway in the dark.

References

  • Andersson, 2008. Catalysis and regulation in Rubisco. Journal of Experimental Botany, vol. 59, pp. 1555-1568
  • Bonacci, Walter, Poh K. Teng, Bruno Afonso, Henrike Niederholtmeyer, Patricia Grob, Pamela A. Silver, und David F. Savage. „Modularity of a Carbon-Fixing Protein Organelle“. Proceedings of the National Academy of Sciences 109, Nr. 2 (1. Oktober 2012): 478–83. doi:10.1073/pnas.1108557109.
  • Jordan, Ogren 1981. Species variation in the specifity of ribulose biphosphate carboxylase/oxygenase. Nature, vol. 291, pp. 513-515
  • Mann, 1999. Genetic Engineers Aim to Soup up Crop Photosynthesis. Science, vol. 283, pp. 314-316
  • Parikh et al., 2006. Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E. coli. Protein Engineering, Design & Selection, vol. 19, pp. 113-119
  • Rosenthal et al., 2011. Overexpressing the C(3) photosynthesis cycle enzyme sedoheptulose 1,7-bisphosphatase improves photosynthetic carbon gain and yield under fully open air CO(2) fumigation (FACE). BMC Plant Biol., vol. 11, pp. 123
  • Spreitzer, Salvucci, 2002. RUBISCO: Structure, Regulatory Interactions, and Possibilities for a Better Enzyme. Annu. Rev. Plant Biol., vol. 53, pp. 449-475
  • Stolzenberger et al., 2013. Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphate from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus. Journal of Bacteriology, Vol. 195, pp. 5112-5122
  • Hügler, Sievert, 2011. Beyond the Calvin Cycle: Autotrophic Carbon Fixation in the Ocean. Annual Review of Marine Science Vol. 3, pp. 261-289
  • Hariharan et al., 1998. Purification and characterization of phosphoribulokinase from the marine chromophytic alga Heterosigma carterae. Plant Physiol. Vol. 117, pp. 321-329
  • Wadano et al., 1995. Purification and characterization of phosphoribulokinase from the cyanobacterium Synechococcus PCC7942. Plant Cell Physiol. Vol. 36, pp. 1381-1385
  • Rumpho et al., 2009. Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica. Molecular Plant. Vol. 2, pp. 1384-1396
  • Michelet, L. et al., 2013.Redox regulation of the Calvin-Benson cycle: something old, something new. Front Plant Sci, vol. 4.

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