Wednesday, July 22, 2015

Can we genetically engineer Rubisco to feed the world?

Today’s atmosphere is brought to you by Rubisco.
Fine makers of oxygen since 3.5 billion B.C.
By Rich Feldenberg

If you happen to peak outside on a nice sunny summers day to admire the green grass, shady trees, and pleasant bushes, your field of view is, in actuality, filled with Rubisco, busily helping the plants do their special thing of making sugar and churning out oxygen.  Rubisco is by far the most abundant enzyme on the earth and accounts for 30%, or more of the protein found in the green leaves of plants.  Without it there would be no oxygen producing photosynthesis, so if you’re a fan of breathing then you’re probably going to be happy to learn about Rubisco.  And, If you’re thinking to yourself, “if there is that much of it in the world it must be doing something important”, congratulations, you’d be right!  

There are three things I’d like to point out about Rubisco. One is that Rubisco is freaking amazing.  It is an awesome protein with interesting molecular properties, catalyzing fascinating chemistry, and dating back to some of the earliest life on earth.  The second thing is that as amazing as Rubisco is, it is incredibly poorly mesigned (mesigned is my word meaning designed by natural selection).  Rubisco is horribly inefficient and slow, and it’s amazing it hasn’t been fired from it’s post and replaced with a new, younger, more hip version.  And finally, Rubisco could, in principle, be engineered to be much better, possibly increasing crop yields to feed an increasing global population, and removing CO2 from the air to combat global warming.  Let's tackle each of these points.

Point One:, Rubisco is amazing.  Without it life on earth would likely still be living as simple single celled mats of slime on the ocean floor in a oxygen free world.  Rubisco is the abbreviated form for the formal name of the enzyme Rubulose-1,5-bisphosphate carboxylase/oxygenase.  Yeah, that’s why Rubisco (rhymes with San Francisco) rolls off the tongue so much nicer, and is way easier to say three times fast.  This enzyme goes way back to the good old days when singled cell organisms ruled the world, and appears to have a common origin in all three of the major kingdoms of living things -bacteria, archaea, and eukaryotes- indicating a very early origin sometime around the time of the Last Universal Common Ancestor (LUCA).  It appears to have arisen even before the evolution of oxygen producing photosynthesis.  There are, by the way, other types of photosynthesis that do not produce oxygen as a byproduct - so called anaerobic photosynthesis- which are less efficient than the oxygen producing types.  Based on the study of related proteins, known as Rubisco-like proteins (RLPs), Rubisco may have evolved from RLPs that performed other enzymatic functions before it was eventually modified to it’s modern role in photosynthesis.  

Rubisco is a big protein, that is itself composed of two main types of subunits - the large subunit (L) and the small subunit (S).  There are three major forms of Rubisco, but in most plants and algae, the Rubisco is composed of a combination of eight L-subunits and eight S-subunits.  Rubisco catalyzes the first step in the photosynthetic process taking CO2 and making it react with the compound ribulose 1,5-bisphosphate (RuBP).  That is way the Rubisco enzyme is named Rubulose-1,5-bisphosphate carboxylase/oxygenase.  In one of it’s reactions, it is carboxylating the substrate RuBP.  This leads to the formation of two molecules of phosphoglyceric acid (PGA), that then go through a metabolic pathway called the Calvin cycle.  The PGA products eventually go on to other metabolic pathways, and the result is sweet sweet sugar!

Space filling model of the Rubisco protein structure 

The L subunit has the active site with a critical lysine residue for binding CO2 It actually takes two CO2 molecules to get things going.  The first CO2 molecule is used just as an activator for the enzyme’s active site, but isn’t used in the carboxylation reaction.  The second CO2 molecule is what is used to react with RuBP, and it is this carbon that is added onto the molecule.  The O2 that is eventually released at the end of photosynthesis does not come from the CO2 but is taken from water, which is also necessary in the reaction.   The genes for the L subunit of Rubisco are found in the chloroplasts, tiny organelles within the cells, where Rubisco is conducting its important job.  The S subunit is more of a stabilizing part of the protein and its gene is located in the nucleus, and once the protein is made, needs to be shuttled into the chloroplasts.

Also necessary for the enzyme to function is the presence of an ion of Mg+2 ,which acts to stabilize the activation site.  This process allows one CO2 molecule, along with a molecule of H2O to become incorporated in RuBP.   There is a whole lot of Rubisco in the green leaves of plant to carry out this important chemical reaction.   As we said above, about 30% or more of the protein in the leaves of plants is in the form of Rubisco, so it therefore accounts for a huge amount of nitrogen stored in the biosphere, since proteins contain nitrogen as part of their structure.  

Point Two: Rubisco is so mind numbingly inefficient I am almost embarrassed for our plant cousins.  It turns out that the carboxylase function of Rubisco (you know the really important thing it does by taking CO2 from the air and attaching it to RuBP to begin the process of making carbohydrate) is not the only reaction it performs.  In fact, it’s very name -the unabbreviated one that is- tells you right off that it it also is an oxygenase.  That is the carboxylase/oxygenase last portion of the name.  This means that Rubisco is not terribly selective for CO2, but can also react at the activation site with a molecule of molecular oxygen (O2), which has some chemically similar properties.  This leads to a horribly counterproductive metabolic pathway called photorespiration.  In other word, it is not very selective, and is so nearsighted that it may grab onto an O2 as easily as a CO2.  Normally about 25% of the reactions that Rubisco is catalyzing are with oxygen going down the photorespiration pathway.  Also, keep in mind that in the atmosphere today, and for at least the last billion years, the concentration of O2 has been way in excess of that of CO2 The atmosphere these days is 21% O2 and only 0.04% CO2, so that makes it even more difficult for poor little Rubisco to discriminate effectively.  

Ribulose 1,5-bisphosphate (RuBP)

Photorespiration leads to RuBP being converted into one molecule of PGA and one molecule of 2-phosphoglycolate.  This doesn’t lead to carbohydrate production.  Even worse this uses energy in the form of ATP and released CO2 into the air.  Totally wrong if you want to store energy from the sun in the form of yummy sugar molecules.  So we can clearly say that Rubisco has a poor affinity for CO2 An enzyme’s affinity for its substrate is measured by a characteristic called Km, and Rubisco’s Km is kind of wimpy.   This relative non-selectivity may be a reflection of the world in which Rubisco first evolved.  At that time the concentration of CO2 in the atmosphere would have been much higher and the concentration of O2 would have been extremely low since photosynthesis was just getting started.  Rubisco probably didn’t need to be too selective since O2 was just a trace gas back then.

The selectivity of Rubisco for CO2 over O2 is affected by temperature.  Warmer temperatures decrease the selectivity making Rubisco even more inefficient.  That can be a problem for plants in a hot dry climate.  Also a change in the amounts of CO2 to O2 with respect to each other will influence the enzyme efficiency.  These two factors may become a significant concern in a world of global climate change where the both the temperature and concentration of CO2 are on the increase.  How this could affect the world’s already insufficient food supplies will have to be seen.

Besides it’s affinity for reacting with a substrate, another characteristic of an enzyme is it’s rate of reaction called the Vmax Guess what, Rubisco’s Vmax also really sucks.  Probably not what you would expect for an enzyme that is the most abundant in the world.  Where as most enzymes catalyze thousands of reactions per second, Rubisco is only able to catalyze about 10 reactions per second.  Now, I hate to sound so judgmental, but that is really pathetic!  It is certainly possible that Rubisco was never able to evolve to be more efficient due to constraints on its structure once it became vital to the plant way of life.  Any alteration in the critical active site may have affected too many other protein-protein interactions necessary for normal function, and so never took place.  Alternatively, there may be some advantages to photorespiration, after all, so that completely shutting down that pathway would, likewise, be detrimental to growth.  There seems to be a trade off between having organisms who’s Rubisco has good affinity for CO2 (Km) and those who’s Rubisco has a fast reaction rate (Vmax).  It’s a case of, you can’t have your cake and eat it too.  If you favor one quality then you suffer in the other.

Plants have come up with a few smart ways of helping to boost the efficiency of Rubisco.  One way is to attempt to concentrate the amount of CO2 around the enzyme.  C4 plants do this by adding the carbon from a CO2 molecule to phosphenolpyruvate (PEP), then through a series of chemical reactions, the organic compound malate is produced.  The malate is shuttled to the plant cells that contain Rubisco and the CO2 is removed.  This concentrates the CO2 in the vicinity of Rubisco so it can act more efficiently.  The waste in energy to produce the malate is more than made up for by the better efficiency of the Rubisco in C4 plants due to this CO2 concentrating ability.  C4 plants are a more recent evolutionary development, but only represents about 3% of land plants.  They are well suited for living in desert conditions where C3 plants would not be able to photosynthesize effectively and would rapidly lose too much water.  C3 plants to well in moderate climates with only moderate sun light.  The lower temperatures helps to improve Rubisco efficiency at utilizing CO2 over O2.  

C4 plants are therefore more efficient, especially in warm dry climates.  CAM (Crassulacean acid metabolism) plants close their stomata in the day to prevent fluid loss and open them at night to allow diffusion of CO2 into the leaves, where it is stored in malate.  During the day the CO2 is again removed from the malate so it can be used by Rubisco to make carbohydrate.  CAM plants can be either land or aquatic.  

The C4 plant, Maize, busily concentrating CO2 to boost Rubisco efficiency

Point Three: Perhaps we can make a better Rubisco, one that can select COover O2 more effectively, and react more quickly.   Nature has had billions of years to figure this out, so maybe its our turn now to design a Rubisco that can be improved in a variety of different ways.  This might be accomplished by artificial selection or genetic engineering - to produce a Genetically modified organism (GMO) with the desirable traits we choose.  In fact, there is a great deal of research looking into possible ways to improve Rubisco, but so far progress seems to have been rather modest.  A super Rubisco could in theory produce more carbohydrate under warmer, drier, and lower light conditions, decrease the amount of nutrient nitrogen necessary for plant growth, and remove more CO2 from the atmosphere, and release more O2.  This could be vital to consider for a growing global population that is outstripping its food resources and heading towards potential disaster due to global warming.  How could it be done?

It is known that red algae has a Rubisco with the highest value yet found for CO2   affinity.  It is nearly 3 times better at discriminating CO2 from O2 than is the Rubisco from crops, like corn.  It may be theoretically possible to engineer crop plants to express the red algae Rubisco.   Other studies have looked to genetically engineering Rubisco by substituting key amino acid residues in critical areas of the enzymes protein structure and observing the effect.  This has resulted in some mild success.  In one study, by switching a particular alanine residue in the L subunit with a asparagine, the affinity was increased by 9%.  Not a huge increase, but potentially a good starting point.  

Other research has focused on speeding up Rubisco’s slow rate of reaction.  One way to accomplish this could be to create a CO2 concentrating mechanism in C3 plants like corn and rice, that is similar to the natural CO2 concentrating mechanisms found in C4 plants.  The ways to make this happen are less clear, but could involve manipulations that would put certain types of COtransporter in the membranes of chloroplasts to help concentrate the gas where it needs to be.  

It should also be noted, that while the intended effects for changes to Rubisco protein would be for the common good of the planet, if we get to the point where such genetically modified plants are possible, it would need to be studied, not only to determine that there are no unintended consequences on the environment, but also that these changes actually result in greater plant growth and yield.  There may be some reasons why photorespiration is allowed to occur at the high rates it does.  One theory is that this is a protective mechanism for the plant so that in intense light conditions energy overload does not occur that could result in oxidative damage to the plant.  There may be a certain limiting factor where carboxylation can be maximized to a certain degree, but once you cross some threshold it actually becomes detrimental to the organism.  

There is no doubt that Rubisco is a curious and fascinating protein, and one on which our lives, and continued survival, are completely dependent.  It is certainly worthy of our admiration for its important and ancient role in maintaining earths biosphere.  There seems to be much more we need to understand about its biochemistry before we can tell if it will be a tool we can utilize to improve and protect our planet.  It could also potentially be altered in algae or cyanobacteria to terraform other planets like Mars, which although it has an extremely thin atmosphere, does have an abundance of CO2 over O2 If we eventually discover life on other world that have evolved some form of photosynthesis, it will be interesting to learn what proteins or other methods they came up with to catalyze the carboxylation reaction that Rubisco serves for us here on earth.

1. M. A. J. Parry, et al., “Manipulation of Rubisco: the amount, activity, function, and regulation”. Journal of Experimental Botany, Vol 54, No. 386,  pp. 1321-1333, May 2003.

2. Spencer M. Whitney, et. al., “Advancing our understanding and capacity to engineer natures CO2-sequestering enzyme, Rubisco”, Plant  Physiology, Vol. 155, pp. 27-35, Jan. 2011.

3. Wikipedia article on Rubisco:

4. Wikipedia article on Photorespiration:

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