RuBisCO
Ribulose-1,5-bisphosphate carboxylase oxygenase | |||||||||
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ExPASy NiceZyme view | | ||||||||
KEGG | KEGG entry | ||||||||
MetaCyc | metabolic pathway | ||||||||
PRIAM | profile | ||||||||
PDB structures | RCSB PDB PDBe PDBsum | ||||||||
Gene Ontology | AmiGO / QuickGO | ||||||||
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Ribulose-1,5-bisphosphate carboxylase/oxygenase, commonly known by the abbreviations RuBisCo, rubisco,
Alternative carbon fixation pathways
RuBisCO is important
Structure
In plants,
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).[12] Mg2+ operates by driving deprotonation of the Lys210 residue, causing the Lys residue to rotate by 120 degrees to the trans conformer, decreasing the distance between the nitrogen of Lys and the carbon of CO2. The close proximity allows for the formation of a covalent bond, resulting in the carbamate.[13] Mg2+ is first enabled to bind to the active site by the rotation of His335 to an alternate conformation. Mg2+ is then coordinated by the His residues of the active site (His300, His302, His335), and is partially neutralized by the coordination of three water molecules and their conversion to −OH.[13] This coordination results in an unstable complex, but produces a favorable environment for the binding of Mg2+. 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 chloroplast) increases in the light. The role of changing pH and magnesium ion levels in the regulation of RuBisCO enzyme activity is discussed below. Once the carbamate is formed, His335 finalizes the activation by returning to its initial position through thermal fluctuation.[13]
Enzymatic activity
RuBisCO is one of many enzymes in the
Substrates
Binding RuBP
Carbamylation of the ε-amino group of Lys210 is stabilized by coordination with the Mg2+.[19] This reaction involves binding of the carboxylate termini of Asp203 and Glu204 to the Mg2+ ion. The substrate RuBP binds Mg2+ displacing two of the three aquo ligands.[14][20][21]
Enolisation
Carboxylation
Carboxylation of the 2,3-enediolate results in the intermediate 3-keto-2-carboxyarabinitol-1,5-bisphosphate and Lys334 is positioned to facilitate the addition of the CO2 substrate as it replaces the third Mg2+-coordinated water molecule and add directly to the enediol. No Michaelis complex is formed in this process.[14][22] Hydration of this ketone results in an additional hydroxy group on C3, forming a gem-diol intermediate.[20][23] Carboxylation and hydration have been proposed as either a single concerted step[20] or as two sequential steps.[23] Concerted mechanism is supported by the proximity of the water molecule to C3 of RuBP in multiple crystal structures. Within the spinach structure, other residues are well placed to aid in the hydration step as they are within hydrogen bonding distance of the water molecule.[14]
C-C bond cleavage
The gem-diol intermediate cleaves at the C2-C3 bond to form one molecule of glycerate-3-phosphate and a negatively charged carboxylate.[14] Stereo specific protonation of C2 of this carbanion results in another molecule of glycerate-3-phosphate. This step is thought to be facilitated by Lys175 or potentially the carbamylated Lys210.[14]
Products
When carbon dioxide is the substrate, the product of the carboxylase reaction is an unstable six-carbon phosphorylated intermediate known as 3-keto-2-carboxyarabinitol-1,5-bisphosphate, which decays rapidly into two molecules of
When molecular oxygen is the substrate, the products of the oxygenase reaction are
Rubisco side activities can lead to useless or inhibitory by-products. Important inhibitory by-products include xylulose 1,5-bisphosphate and glycero-2,3-pentodiulose 1,5-bisphosphate, both caused by "misfires" halfway in the enolisation-carboxylation reaction. In higher plants, this process causes RuBisCO self-inhibition, which can be triggered by saturating CO2 and RuBP concentrations and solved by Rubisco activase (see below).[24]
Rate of enzymatic activity
Some enzymes can carry out thousands of chemical reactions each second. However, RuBisCO is slow, fixing only 3-10 carbon dioxide molecules each second per molecule of enzyme.[25] The reaction catalyzed by RuBisCO is, thus, the primary rate-limiting factor of the Calvin cycle during the day. Nevertheless, under most conditions, and when light is not otherwise limiting photosynthesis, the speed of RuBisCO responds positively to increasing carbon dioxide concentration.
RuBisCO is usually only active during the day, as ribulose 1,5-bisphosphate is not regenerated in the dark. This is 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 other ways:
By ions
Upon illumination of the chloroplasts, the
By RuBisCO activase
In plants and some algae, another enzyme, RuBisCO activase (Rca, GO:0046863, P10896), is required to allow the rapid formation of the critical
By 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 substrate.[32]
By phosphate
In cyanobacteria, inorganic phosphate (Pi) also 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. In this way, activation of bacterial RuBisCO might be particularly sensitive to Pi levels, which might cause it to act in a similar way to how RuBisCO activase functions in higher plants.[33]
By carbon dioxide
Since carbon dioxide and oxygen
Genetic engineering
Since RuBisCO is often rate-limiting for photosynthesis in plants, it may be possible to improve
Mutagenesis in plants
In general, site-directed mutagenesis of RuBisCO has been mostly unsuccessful,[38] though mutated forms of the protein have been achieved in tobacco plants with subunit C4 species,[41] and a RuBisCO with more C4-like kinetic characteristics have been attained in rice via nuclear transformation.[42] Robust and reliable engineering for yield of RuBisCO and other enzymes in the C3 cycle was shown to be possible,[43] and it was first achieved in 2019 through a synthetic biology approach.[37]
One avenue is to introduce RuBisCO variants with naturally high specificity values such as the ones from the
A recent theory explores the trade-off between the relative specificity (i.e., ability to favour CO2 fixation over O2 incorporation, which leads to the energy-wasteful process of photorespiration) and the rate at which product is formed. The authors conclude that RuBisCO may actually have evolved to reach a point of 'near-perfection' in many plants (with widely varying substrate availabilities and environmental conditions), reaching a compromise between specificity and reaction rate.[47] It has been also suggested that the oxygenase reaction of RuBisCO prevents CO2 depletion near its active sites and provides the maintenance of the chloroplast redox state.[48]
Since photosynthesis is the single most effective natural regulator of
Expression in bacterial hosts
There currently are very few effective methods for expressing functional plant Rubisco in bacterial hosts for genetic manipulation studies. This is largely due to Rubisco's requirement of complex cellular machinery for its biogenesis and metabolic maintenance including the nuclear-encoded RbcS subunits, which are typically imported into chloroplasts as unfolded proteins.[50][51] Furthermore, sufficient expression and interaction with Rubisco activase are major challenges as well.[39] One successful method for expression of Rubisco in E. coli involves the co-expression of multiple chloroplast chaperones, though this has only been shown for Arabidopsis thaliana Rubisco.[52]
Depletion in proteomic studies
Due to its high abundance in plants (generally 40% of the total protein content), RuBisCO often impedes analysis of important signaling proteins such as transcription factors, kinases, and regulatory proteins found in lower abundance (10-100 molecules per cell) within plants.[53] For example, using mass spectrometry on plant protein mixtures would result in multiple intense RuBisCO subunit peaks that interfere and hide those of other proteins.
Recently, one efficient method for precipitating out RuBisCO involves the usage of protamine sulfate solution.[54] Other existing methods for depleting RuBisCO and studying lower abundance proteins include fractionation techniques with calcium and phytate,[55] gel electrophoresis with polyethylene glycol,[56][57] affinity chromatography,[58][59] and aggregation using DTT,[60] though these methods are more time-consuming and less efficient when compared to protamine sulfate precipitation.[53]
Evolution of RuBisCO
Phylogenetic studies
The chloroplast gene rbcL, which codes for the large subunit of RuBisCO has been widely used as an appropriate locus for analysis of phylogenetics in plant taxonomy.[61]
Origin
external image}} pointing to the MotM and Erb 2018 pics. . (March 2022) |
Non-carbon-fixing proteins similar to RuBisCO, termed RuBisCO-like proteins (RLPs), are also found in the wild in organisms as common as Bacillus subtilis. This bacterium has a rbcL-like protein with a 2,3-diketo-5-methylthiopentyl-1-phosphate enolase function, part of the methionine salvage pathway.[62] Later identifications found functionally divergent examples dispersed all over bacteria and archaea, as well as transitionary enzymes performing both RLP-type enolase and RuBisCO functions. It is now believed that the current RuBisCO evolved from a dimeric RLP ancestor, acquiring its carboxylase function first before further oligomerizing and then recruiting the small subunit to form the familiar modern enzyme.[15] The small subunit probably first evolved in anaerobic and thermophilic organisms, where it enabled RuBisCO to catalyze its reaction at higher temperatures.[63] In addition to its effect on stabilizing catalysis, it enabled the evolution of higher specificities for CO2 over O2 by modulating the effect that substitutions within RuBisCO have on enzymatic function. Substitutions that do not have an effect without the small subunit suddenly become beneficial when it is bound. Furthermore, the small subunit enabled the accumulation of substitutions that are only tolerated in its presence. Accumulation of such substitutions leads to a strict dependence on the small subunit, which is observed in extant Rubiscos that bind a small subunit.
C4
With the mass convergent evolution of the
History of the term
The term "RuBisCO" was coined humorously in 1979, by
The capitalization of the name has been long debated. It can be capitalized for each letter of the full name (Ribulose-1,5 bisphosphate carboxylase/oxygenase), but it has also been argued that is should all be in lower case (rubisco), similar to other terms like scuba or laser.[1]
See also
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References
- bacterial RuBisCO is shown in the Protein Data Bank "Molecule of the Month" #11.[11]
- ^ S2CID 53092349.
- PMID 16663341.
- PMID 16666327.
- ^ Back to the future of photosynthesis: Resurrecting billon-year-old enzymes reveals how photosynthesis adapted to the rise of oxygen., News from the Max Planck Society, October 13, 2022
- ISBN 978-0-87893-106-4.
, one of the subunits of ribulose bisphosphate carboxylase (rubisco) is encoded by chloroplast DNA. Rubisco is the critical enzyme that catalyzes the addition of CO2 to ribulose-1,5-bisphosphate during the Calvin cycle. It is also thought to be the single most abundant protein on Earth, so it is noteworthy that one of its subunits is encoded by the chloroplast genome.
- ^ PMID 15067115.
(Rubisco) is the most prevalent enzyme on this planet, accounting for 30–50% of total soluble protein in the chloroplast;
- ^ PMID 17975207.
- PMID 29594130.
- PMID 11401297.
- ISBN 978-0-7167-3051-4.
Figure 20.3. Structure of Rubisco.] (Color-coded ribbon diagram)
- .
- ^ Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell JE (2000). "Molecular Cell Biology" (4th ed.). New York: W. H. Freeman & Co. Figure 16-48 shows a structural model of the active site, including the involvement of magnesium.
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- .
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- ^ Timmer J (7 December 2017). "We may now be able to engineer the most important lousy enzyme on the planet". Ars Technica. Retrieved 5 January 2019.
- ^ Timmer J (3 January 2019). "Fixing photosynthesis by engineering it to recycle a toxic mistake". Ars Technica. Retrieved 5 January 2019.
- ^ PMID 30606819.
- ^ .
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- S2CID 39767233.
Further reading
- Marcus Y, Altman-Gueta H, Finkler A, Gurevitz M (June 2005). "Mutagenesis at two distinct phosphate-binding sites unravels their differential roles in regulation of Rubisco activation and catalysis". Journal of Bacteriology. 187 (12): 4222–4228. PMID 15937184.
- Sugawara H, Yamamoto H, Shibata N, Inoue T, Okada S, Miyake C, et al. (May 1999). "Crystal structure of carboxylase reaction-oriented ribulose 1, 5-bisphosphate carboxylase/oxygenase from a thermophilic red alga, Galdieria partita". The Journal of Biological Chemistry. 274 (22): 15655–15661. PMID 10336462.
External links
- Gerritsen VB (September 2003). "The Plant Kingdom's sloth". Protein Spotlight. Swiss Institute of Bioinformatics (SIB).
Rubisco plods along at a mere three molecules per second... To bypass such slothfulness, plants synthesize a gross amount of Rubisco, sometimes up to 50% of their total protein content!