RuBisCO

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Ribulose-1,5-bisphosphate carboxylase/oxygenase, most commonly known by the shorter name RuBisCO, is an enzyme (Template:EC number) that is used in the Calvin cycle to catalyze the first major step of carbon fixation, a process by which the atoms of atmospheric carbon dioxide are made available to organisms in the form of energy-rich molecules such as sucrose. RuBisCO catalyzes either the carboxylation or oxygenation of ribulose-1,5-bisphosphate (also known as RuBP) with carbon dioxide or oxygen.

RuBisCO is very important in terms of biological impact because it catalyzes the most commonly used chemical reaction by which inorganic carbon enters the biosphere. RuBisCO is apparently the most abundant protein in leaves, and it may be the most abundant protein on EarthTemplate:Ref. Given its important role in the biosphere, there are currently efforts to "improve on nature" and genetically engineer crop plants so as to contain more efficient RuBisCO (see below).

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Structure

In plants, algae, cyanobacteria, and phototropic and chemoautotropic proteobacteria the enzyme usually consists of two types of protein subunit, called the large chain (L, about 55,000 Da) and the small chain (S, about 13,000 Da)Template:Ref. The enzymatically active substrate (ribulose 1,5-bisphosphate) binding sites are located in the large chains that form dimers as shown in Figure 1 (above, right) in which amino acids from each large chain contribute to the binding sites. A total of four large chain dimers and eight small chains assemble into a larger complex of about 540,000 DaTemplate:Ref. In some proteobacteria and dinofagellates, enzymes consisting of only large subunits have been found Template:Ref.

Magnesium ions (Mg2+) are needed for enzymatic activity. Correct positioning of Mg2+ in the active site of the enzyme involves addition of an "activating" carbon dioxide molecule (CO2) to a lysine in the active site (forming a carbamate)Template:Ref. Formation of the carbamate is favored by an alkaline pH. The pH and the concentration of magnesium ions in the fluid compartment (in plants, the stroma of the chloroplastTemplate:Ref) increases in the light. The role of changing pH and magnesium ion levels in the regulation of RuBisCO enzyme activity is discussed below.

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Enzymatic Activity

Image:Calvin-cycle3.png As shown in Figure 2 (left), RuBisCO is one of many enzymes in the Calvin cycle.
Substrates. During carbon fixation, the substrate molecules for RuBisCO are ribulose 1,5-bisphosphate, carbon dioxide (distinct from the "activating" carbon dioxide) and water Template:Ref. RuBisCO can also allow a reaction to occur with molecular oxygen (O2) instead of carbon dioxide (CO2).
Products. When carbon dioxide is the substrate, the product of the carboxylase reaction is 3-phosphoglycerate. The 3-phosphoglycerate can be used to produce larger molecules such as glucose. When molecular oxygen is the substrate, the products of the oxygenase reaction are phosphoglycolate and 3-phosphoglycerate. Phosphoglycolate initiates a sequence of reactions called photorespiration which involves enzymes located in the mitochondria and peroxisomes. In this process, two molecules of phosphoglycolate are converted to one molecule of carbon dioxide and one molecule of 3-phosphoglycerate, which can reenter the Calvin cycle. Some of the phosphoglycolate entering this pathway can be retained by plants to produce other molecules such as glycine. At air levels of carbon dioxide and oxygen, the ratio of the reactions is about 4 to 1, which results in a net carbon dioxide fixation of only 3.5. Thus the inability of the enzyme to prevent the reaction with oxygen greatly reduces the photosynthetic potential of many plants. Some plants and many algae and photosynthetic bacteria have overcome this limitation by devising means to increase the concentration of carbon dioxide around the enzyme, including C4 carbon fixation, crassulacean acid metabolism and using pyrenoid.

Rate of enzymatic activity. Some enzymes can carry out thousands of chemical reactions each second. RuBisCO is slow, being able to "fix" only a few carbon dioxide molecules each second. Under most conditions and when light is not otherwise limiting photosynthesis, RuBisCO is the primary rate-limiting enzyme of the Calvin cycle.

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Regulation of its Enzymatic Activity

RuBisCO is usually only active during the day because ribulose 1,5-bisphosphate is not being produced in the dark due to the regulation of several other enzymes in the Calvin cycle. In addition, the activity of Rubisco is coordinated with that of the other enzymes of the Calvin cycle in several ways:


  1. Regulation by ions. Upon illumination of the chloroplasts, the pH of the stroma rises from 7.0 to 8.0 because of the proton (hydrogen ion, H+) gradient created across the thylakoid membraneTemplate:Ref. At the same time, magnesium ions (Mg2+) move out of the thylakoids, increasing the concentration of magnesium in the stroma of the chloroplasts. RuBisCO has a high optimal pH (can be >9.0, depending on the magnesium ion concentration) and thus becomes "activated" by the addition of carbon dioxide and magnesioum to the active sites as described above.
  2. Regulation by activase. In plants and some algae, another enzyme, RuBisCO activaseTemplate:Ref is required to allow the rapid formation of the critical carbamate in the active site of RuBisCOTemplate:Ref. Activase is required because the ribulose 1,5-bisphosphate substrate binds more strongly to the active sites lacking the carbamate and markedly slows down the "activation" process. In the light, RuBisCO activase promotes the release of the inhibitory ribulose 1,5-bisphosphate from the catalytic sites. Activase is also required in some plants (e.g. tobacco and many beans) because in darkness, RuBisCO is inhibited by a competitive inhibitor synthesized by these plants, a substrate analog 2-Carboxy-D-arabitinol 1-phosphate (CA1P)Template:Ref. CA1P binds tightly to the active site of carbamylated RuBisCO and inhibits catalytic activity. In the light, RuBisCO activase also promotes the release of CA1P from the catalytic sites. After the CA1P is released from RuBisCO, it is rapidly converted to a non-inhibitory form by a light-activated CA1P-phosphatase. Finally, once every several hundred reactions, the normal reactions with carbon dioxide or oxygen are not completed and other inhibitory substrate analogs are formed in the active site. Once again, RuBisCO activase can promote the release of these analogs from the catalytic sites and maintain the enzyme in a catalytically active form. The properties of activase limit the photosynthetic potential of plants at high temperatures Template:Ref. CA1P has also been shown to keep Rubisco in a conformation that is protected from proteolysisTemplate:Ref.
  3. Regulation by ATP/ADP and stromal reduction/oxidation state through the activase. The removal of the inhibitory RuBP, CA1P, and the other inhibitory substrate analogs by activase requires the consumption of ATP. This reaction is inhibited by the presence of ADP and thus activase activity depends on the ratio of these compounds in the chloroplast stroma. Furthermore in most plants, the sensitivity of activase to the ratio of ATP/ADP is modified by the stromal reduction/oxidation (redox) state through another small regulatory protein, thioredoxin. In this manner, the activity of activase and the activation state of Rubisco can be modulated in response to light intensity and thus the rate of formation of the ribulose 1,5-bisphosphate substrateTemplate:Ref.
  4. Regulation by phosphate. Inorganic phosphate (Pi) participates in the co-ordinated regulation of photosynthesis. Pi binds to the RuBisCO active site and to another site on the large chain where it can influence transitions between activated and less active conformations of the enzyme. Activation of bacterial RuBisCO might be particularly sensitive to Pi levelsTemplate:Ref.
  5. Regulation by carbon dioxide. Since carbon dioxide and oxygen compete at the active site of RuBisCO, carbon fixation by RuBisCO can be enhanced by increasing the carbon dioxide level in the compartment containing RuBisCO (chloroplast stroma). Several times during the evolution of plants, mechanisms have evolved for increasing the level of carbon dioxide in the stroma (see C4 carbon fixation). The use of oxygen as a substrate is an apparently-puzzling process, since it seems to throw away captured energy. However it may be a mechanism for preventing overload during periods of high light flux. C4 plants use the enzyme PEP carboxylase initially, which has a higher affinity for CO2. The process first makes a 4-carbon intermediate compound; hence the name C4 plants.

Crassulacean acid metabolism (CAM) plants keep their stomata (on the underside of the leaf) closed during the day, which conserves water but prevents photosynthesis, which requires CO2 to pass by gas exchange through these openings. Evaporation through the upper side of a leaf is prevented by a layer of wax.

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Genetic engineering

Since RuBisCO is often rate limiting for photosynthesis in plants, it may be possible to improve photosynthetic efficiency by modifying RuBisCO genes in plants to increase its catalytic activity and/or decrease the rate of the oxygenation activityTemplate:Ref. Approaches that have begun to be investigated include expressing RuBisCO genes from one organism in another organism, increasing the level of expression of RuBisCO subunits, expressing RuBisCO small chains from the chloroplast DNA, and altering RuBisCO genes so as to try to increase specificity for carbon dioxide or otherwise increase the rate of carbon fixationTemplate:Ref.

One particularly interesting avenue is to introduce rubiscos with naturally high specificity values such as the ones from the red algae Galdieria partita into plants. This would be expected to improve the photosynthetic efficiency of crop plants Template:Ref. Important advances in this area include the replacement of the tobacco enzyme with that of the purple photosynthetic bacterium Rhodospirillum rubrum Template:Ref.

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References

Image:RuBisCOL2S2.png Image:RuBisCO.jpg

  1. Template:Note The Cell—A Molecular Approach. 2nd ed. by Geoffrey M. Cooper, published by Sinauer Associates, Inc. (2000) Sunderland (MA). Online textbook. The Cooper text suggests that RuBisCO is the most abundant protein on Earth (Chapter 10, The Chloroplast Genome). A recent article by Dhingra et al, suggests that RuBisCO accounts for 30–50% of total soluble protein in chloroplasts (full text article online: Template:Entrez Pubmed).
  2. Template:Note The large chain gene is part of the chloroplast DNA molecule in plants (Entrez GeneID: 3052726). There are typically several related small chain genes in the nucleus of plant cells and the small chains are imported to the stromal compartment of chloroplasts from the cytosol by crossing the outer chloroplast membrane (full text article online: Template:Entrez Pubmed). Arabidopsis thaliana has four RuBisCO small chain genes (see: Template:Entrez Pubmed). The pattern of how large chains and small chains assemble is illustrated in Figure 3 (right).
  3. Template:Note Biochemistry by Jeremy M. Berg, John L. Tymoczko, and Lubert Stryer. Published by W. H. Freeman and Co. (2002) New York. Online textbook. Figure 20 in the Stryer textbook shows a color-coded ribbon diagram of the structural components of eukaryotic RuBisCO. Figure 1 (on this page, near top) shows another view of the structure.
  4. Template:Note The structure of RuBisCO from the photosynthetic bacterium Rhodospirillum rubrum has been determined by X-ray crystallography, see: Template:Protein Data Bank. A comparison of the structures of eukaryotic and bacterial RuBisCO is shown in the Protein Data Bank feature article on Rubisco.
  5. Template:Note Molecular Cell Biology, 4th edition, by Harvey Lodish, Arnold Berk, S. Lawrence Zipursky, Paul Matsudaira, David Baltimore and James E. Darnell. Published by W. H. Freeman & Co. (2000) New York. Online textbook. Figure 16-48 shows a structural model of the active site, including the involvement of magnesium. The Protein Data Bank feature article on RuBisCO also includes a model of magnesium at the active site.
  6. Template:Note The Lodish textbook describes the localization of RuBisCO to the stromal space of chloroplasts. Figure 17-7 illustrates how RuBisCO small subunits move into the chloroplast stroma and assemble with the large subunits.
  7. Template:Note The chemical reactions catalyzed by RuBisCO are described in the online Biochemistry textbook by Stryer et al.
  8. Template:Note Figure 20.14 in the textbook by Stryer et al. illustrates the light-dependent movement of hydrogen and magnesium ions that are important for Light Regulation of the Calvin Cycle. The movement of protons into thylakoids is driven by light and is fundamental to ATP synthesis in chloroplasts.
  9. Template:Note "Rubisco activase—Rubisco's catalytic chaperone." by A. R. Portis, Jr in Photosynthesis Research (2003), volume 75, pages 11–27. (see: Template:Entrez Pubmed).
  10. Template:Note "Characteristics of photosynthesis in rice plants transformed with an antisense Rubisco activase gene" by S. H. Jin, D. A. Jiang, X. Q. Li and J. W. Sun. Transgenic plants that were genetically engineered to have reduced levels of RuBisCO activase were shown to have reduced photosynthesis (see: Template:Entrez Pubmed).
  11. Template:Note"Incorporation of carbon from photosynthetic products into 2-carboxyarabinitol-1-phosphate and 2-carboxyarabinitol." by P. J. Andralojc, G. W. Dawson, M. A. Parry and A. J. Keys in Biochemical Journal (1994), volume 304, pages 781–6. (full text online: Template:Entrez Pubmed).
  12. Template:Note "Rubisco activase constrains the photosynthetic potential of leaves at high temperature and CO2. by S. J. Crafts-Brandner and M. E. Salvucci in Proceedings of the National Academy of Science USA (2000), volume 97, pages 12937–8. (full text online:Template:Entrez Pubmed).
  13. Template:Note"2'-carboxy-D-arabitinol 1-phosphate protects ribulose 1, 5-bisphosphate carboxylase/oxygenase against proteolytic breakdown" by S. Khan, P. J. Andralojc, P. J. Lea and M. A. Parry in European Journal of Biochemistry (1999), volume 266, pages 840–7. (full text online: Template:Entrez Pubmed).
  14. Template:Note "Light modulation of Rubisco in Arabidopsis requires a capacity for redox regulation of the larger Rubisco activase isoform.." by N. Zhang, R. Kallis, R. G. Ewy, A. R. Portis Jr in Proceedings of the National Academy of Science USA (2002), volume 99, pages 3330–4 (full text online:Template:Entrez Pubmed).
  15. Template:Note "Activation of cyanobacterial RuBP-carboxylase/oxygenase is facilitated by inorganic phosphate via two independent mechanisms." by Yehouda Marcus and Michael Gurevitz in European Journal of Biochemistry (2000), volume 267, pages 5995–6003. (full text online:Template:Entrez Pubmed).
  16. Template:Note "Rubisco: structure, regulatory interactions, and possibilities for a better enzyme." by R. J Spreitzer and M. E. Salvucci in Annual Review of Plant Biology (2003) volume 53, page 449–75 (see: Template:Entrez Pubmed).
  17. Template:Note "Manipulation of Rubisco: the amount, activity, function and regulation." by M. A. Parry, P. J. Andralojc, R. A. Mitchell, P. J. Madgwick and A. J. Keys in Journal of Experimental Botany (2003) volume 54, page 1321–33. (full text online: Template:Entrez Pubmed)
  18. Template:Note "Whitney, S. M. and T. J. Andrews (2001). "Plastome-encoded bacterial ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) supports photosynthesis and growth in tobacco." Proceedings of the National Academy of Sciences of the United States of America 98(25): 14738-14743."
  19. Template:Note "Andrews, T. J. and S. M. Whitney (2003). "Manipulating ribulose bisphosphate carboxylase/oxygenase in the chloroplasts of higher plants." Archives of Biochemistry and Biophysics 414(2): 159-169."
  20. Sugawara H, Yamamoto H, Shibata N, Inoue T, Okada S, Miyake C, Yokota A, Kai Y. Crystal structure of carboxylase reaction-oriented ribulose 1, 5-bisphosphate carboxylase/oxygenase from a thermophilic red alga, Galdieria partita. J Biol Chem 1999; 274:15655–61. Fulltext. PMID 10336462.
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See also

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External links

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