Rubisco is said to be both the most important enzyme on Earth and surprisingly inefficient. Yet an understanding of the reaction by which it fixes CO2 suggests that evolution has made the best of a bad job.
Rubisco is the enzyme in photosynthesis that is responsible for the conversion of inorganic carbon, as CO2, into organic compounds. The demanding initial catalytic step (or carboxylase reaction1) precedes the photosynthetic reduction of reaction products using the energy trapped from sunlight. The acronym Rubisco actually stands for ribulose bisphosphate carboxylase-oxygenase, because the enzyme also has a tendency to confuse O2 for CO2 as its substrate. Rubisco has the reputation of being slow and inefficient, but it is one of life's big successes: globally there is an estimated 5–10 kg Rubisco for every person on Earth, and each year it reacts with 15% of the total pool of atmospheric CO2.
Work to ‘improve’ Rubisco and so increase crop productivity has usually foundered on the catalytic active site, which is highly conserved in different forms of the enzyme and has generally proved to be intractable to genetic manipulation. But a fresh angle on such prospects comes from Tcherkez and colleagues2, writing in the Proceedings of the National Academy of Sciences. They propose an explanation for the reaction mechanism that accounts for the selection of CO2 in preference to O2. The systematic evolution of enzyme kinetic properties seems to have occurred in Rubisco from different organisms, suggesting that Rubisco is well adapted to substrate availability in contrasting habitats.
It is curious that Rubisco should fix CO2 at all, as there is 25 times more O2 than CO2 in solution at 25 °C, and a 500-fold difference between them in gaseous form. Yet only 25% of reactions are oxygenase events at this temperature, and carbon intermediates ‘lost’ to the carbon fixation reactions by oxygenase action are metabolized and partly recovered by the so-called photorespiratory pathway. Catalysis begins with activation of Rubisco by the enzyme Rubisco activase1,3, when first CO2 and then a magnesium ion bind to the active site. The substrate, ribulose bisphosphate, then reacts with these to form an enediol inter-mediate, which engages with either another CO2 or an O2 molecule, either of which must diffuse down a solvent channel to reach the active site. Tcherkez and colleagues' achievement2 is to have produced an explanatory mechanism for the trade-off usually observed between the specificity factor (that is, a ratio indicating selectivity for CO2 over O2, which ranges between 20 and 280; refs 1, 3) and kcat (the rate of enzyme turnover, which varies between two and eight catalytic events per second).
Work leading up to this proposed mechanism4 involved site-directed mutagenesis in tobacco, which had the aim of destabilizing the enzyme active site and altering the reactivity of the enediol intermediate. Changing the binding of an amino-acid residue that encourages the addition of both CO2 and O2 dramatically increased the rate of oxygenase activity.
Such observations3,4 provided the key to the idea2 that in the active site the enediol must be contorted to allow CO2 to attack more readily despite the availability of O2 molecules. The more the enediol mimics the carboxylate end-product, Tcherkez et al.2 conclude, the more difficult it is for the enzyme to free the intermediate from the active site when the reaction is completed. When the specificity factor and selectivity for CO2 are high, the impact on associated kinetic properties is greatest: kcat becomes slower.
So, rather than being inefficient, Rubisco has become highly tuned to match substrate availability. Several other correlates are also explained by this relationship. For instance, Rubisco discriminates more against 13C than against 12C, the two naturally occurring stable isotopes in CO2. But when the specificity factor is high, the 13C reaction intermediate binds more tightly, and so carbon isotope discrimination is higher (that is, less 13C is incorporated); in consequence, the carbon-isotope signals used to reconstruct past climates should perhaps now be re-examined. In contrast, higher ambient temperatures (30–40 °C) reduce the stability of the enediol, and Rubisco oxygenase activity and photorespiration rate increase.
These insights2 into the mechanism of Rubisco catalysis are timely, because progress is being made in identifying the evolutionary origins of a wider range (forms I to IV) of Rubisco-like proteins5. The least effective of these forms have evolved, and now reside, in microorganisms in anaerobic sediments where catalysis does not have to compete with oxygen1. One bacterium can express all three catalytically active forms (I, II and III), and switches between them depending on environmental conditions6. The evolutionary trade-offs observed by Tcherkez et al. are consistent with the occurrence of most of these enzymes. Alternatively, some higher plants and photosynthetic microorganisms have developed mechanisms to suppress oxygenase activity: CO2-concentrating mechanisms are induced either biophysically7 or biochemically8,9. There are systematic changes in Rubisco kinetics1,3, consistent with Tcherkez and colleagues' views, that are thought to have evolved in the past 10 million years in plants related to sugar cane and maize.
Molecular techniques such as ‘directed evolution’ offer the possibility of manipulating part of Rubisco — the large subunit component — to select for catalytic improvements10, allowing access to ‘sequence space’ that is not available to conventional molecular manipulations. A sensible move would be to screen the kinetics of Rubisco enzymes from different plants that inhabit extreme environments1,3. For instance, Rubisco has not been characterized in the so-called CAM plants, which use a form of photosynthesis (crassulacean acid metabolism) adapted for arid conditions. In these species, the enzyme is exposed daily to a range of CO2 concentrations (0.01–2%) equivalent to those occurring throughout much of Earth's history9.
Other research avenues include manipulating the various components of Rubisco1 and cell-specific targeting of chimaeric Rubiscos8. Potential pitfalls here are that the modified Rubisco would not only have to be incorporated and assembled by crop plants, but any improved performance would have to be retained by the plants. Finally, one suggestion is that we should engineer plants that can express two types of Rubisco — each with kinetic properties to take advantage of the degree of shading within a crop canopy11. Such rational design10 would not only offer practical opportunities for the future, but also finally give the lie to the idea that Rubisco is intransigent and inefficient.
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Comparative studies for evaluation of CO2 fixation in the cavity of the Rubisco enzyme using QM, QM/MM and linear-scaling DFT methods
Journal of Molecular Modeling (2013)