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Thursday, July 12, 2007

Fixing Carbon: Building a Better Rubisco

On Not Getting with the Adaptationist Program

As its complete name indicates, ribulose 1,5- bisphosphate carboxylase– oxygenase (Rubisco) catalyzes not only the carboxylation but also the oxygenation of ribulose 1,5-bisphosphate [Fixing Carbon: the Rubisco Reaction]. The two reactions are competitive—CO2 and O2 compete for the same active sites on the Rubisco molecule [Fixing Carbon: the Structure of Rubisco]. The oxygenation reaction produces one molecule of 3-phosphoglycerate and one molecule of 2-phosphoglycolate.

Oxygenation consumes significant amounts of ribulose 1,5- bisphosphate in vivo. Under normal growth conditions, the rate of carboxylation is only about three to four times the rate of the oxygenation reaction. The oxidative pathway consumes NADH and ATP because the products have to be converted back into ribulose 1,5- bisphosphate in order to continue carbon fixation. The light-dependent uptake of O2 catalyzed by Rubisco, followed by the release of CO2 during the subsequent metabolism of 2-phosphoglycolate, is called photorespiration.

The appreciable release of fixed CO2 and the consumption of energy—with no apparent benefit to the plants—arise because of the lack of absolute substrate specificity of Rubisco. This side reaction can greatly limit crop yields. It looks very much as though Rubisco is just an inefficient enzyme that's incapable of distinguishing between two very similar substrates, CO2 and O2. It appears to be an example of a badly "designed" enzyme*, not unlike many others that have significant error rates.

The inefficiency of Rubisco, both in terms or its low reaction rate and its propensity for errors, is why massive amounts of the enzyme are needed in plants. The result is that Rubisco is probably the most abundant enzyme on Earth.

But if nature has done such a bad job of design, can scientists do better? That's the goal of many research labs since improving the efficiency of Rubisco can greatly enhance crop yields, and, incidentally, makes lots of money for the inventors of the genetically modified crops.

Several labs are attempting to genetically modify plants to enhance the carboxylation reaction and suppress the oxygenation reaction. The “perfect” enzyme would have very low oxygenase activity and very efficient carboxylase activity. The kinetic parameters of the carboxylase activity of Rubisco enzymes from several species are listed in the table below (Andrews and Whitney, 2003).

The low catalytic proficiency of the enzyme is indicated by the kcat/KM values. These values should be compared to those of typical enzymes that have values from ten to one thousand times greater. It seems likely that the carboxylase efficiency can be improved 1000-fold by modifying the amino acid side chains in the active site.

The difficult part of the genetic modification is choosing the appropriate amino acid changes. The choice is made easier by a detailed knowledge of the structures of several Rubisco enzymes from different species and by examination of the contacts between amino acid side chains and substrate molecules. Models of the presumed transition states are also important. Additional key residues can be identified by comparing the conservation of amino acid sequences in enzymes from a wide variety of species.

The underlying strategy assumes that evolution has not yet selected for the most well–designed enzyme. This assumption seems reasonable since there are many examples of ongoing evolution in biochemistry. Nevertheless, progress has been slow in spite of the enormous financial rewards.

In addition to intelligently-directed genetic engineering to improve on nature, some groups have relied on a form of artificial evolution to do the job. Here's the abstract of a recent paper (Parikh et al. 2006).
The Calvin Cycle is the primary conduit for the fixation of carbon dioxide into the biosphere; ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the rate-limiting fixation step. Our goal is to direct the evolution of RuBisCO variants with improved kinetic and biophysical properties. The Calvin Cycle was partially reconstructed in Escherichia coli; the engineered strain requires the Synechococcus PCC6301 RuBisCO for growth in minimal media supplemented with a pentose. We randomly mutated the gene encoding the large subunit of RuBisCO (rbcL), co-expressed the resulting library with the small subunit (rbcS) and the Synechococcus PCC7492 phosphoribulokinase (prkA), and selected hypermorphic variants. The RuBisCO variants that evolved during three rounds of random mutagenesis and selection were over-expressed, and exhibited 5-fold improvement in specific activity relative to the wild-type enzyme. These results demonstrate a new strategy for the artificial selection of RuBisCO and other non-native metabolic enzymes.
©Laurence A. Moran and Pearson Prentice Hall 2007

*As you might imagine from our discussion of the adaptationist program, there are many biochemists who are very uncomfortable with the notion of a function that has no adaptive explanation. Textbooks are full of adaptive just-so stories that try to justify the oxygenation reaction. None of them hold up to close scrutiny. The Voet and Voet textbook (Biochemisty) avoids the worst just-so stories by saying, "Although photorespiration has no known metabolic function, the RuBisCOs from a great variety of photosynthetic organisms so far tested all exhibit oxygenase activity. Yet, over the eons, the forces of evolution must have optimized the function of this important enzyme." They then go on to describe two unlikely adaptive explanations of the oxygenation reaction. Note that Don and Judy Voet assume that the very existence of the oxygenation reaction demands an optimization assumption and, consequently, an adaptationist explanation. Accident is not a possibility in their minds.

Andrews, J. T. and Whitney, S. M. (2003) Manipulating ribulose bisphosphate carboxylase/oxygenase in the chloroplasts of higher plants. Arch. Biochem. Biophys. 414:159–169.

Parikh, M.R., Greene, D.N., Woods, K.K., Matsumura, I. (2006) Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E.coli. Protein Eng Des Sel. 19(3):113-9.


Nick (Matzke) said...

Larry -- I agree about adaptationism here.

Here is my question: isn't it true that distinguishing CO2 from O2 is going to be a fundamentally difficult thing to do, chemically speaking, particularly when photosynthesis has sucked CO2 down to 300 ppm and O2 is 210,000 ppm in the atmosphere?

In other words, why does everyone think it should be possible to do very much better with this very difficult chemical process?

(There might actually be a good reason, I'm just asking.)

Larry Moran said...

Nick Matzke asks,

In other words, why does everyone think it should be possible to do very much better with this very difficult chemical process?

Because there are many other kinds of reactions where CO2 is fixed and O2 does not act as a significant competing substrate.

What I don't understand is the perspective of the Intelligent Design Creationists. How do they explain such a sloppy enzyme? Couldn't God have done a better job?

Alex said...

You should iron this out with Professor Pulleyblank, because he mentioned an adaptive explanation for the oxygenation reaction. I'm not sure if he's right or wrong, but blatant contradiction between professors is confusing.

Anonymous said...

A scenario and a question.

The scenario: Carboxylase activity arose or evolved in a time or under conditions where oxygenase activity was not an issue (very low levels of oxygen, say). The nice "fit" of oxygen in the rubisco active site is a coincidence that factored in later in the evolution of life on earth.

The question: Is this an adaptationist rationalization?

Anonymous said...

art, in my opinion you've hit the nail on the head. What has not been discussed here is that the carboxylation reaction is in fact very complex (consisting of five different partial reactions) and that ALL Rubiscos have the same crucial active site residues and catalytic chemistry. Therefore we might not expect evolution to be able to solve the problem.

Anonymous said...

A. J. BLOOM of Davis Ca has an interesting hypothesis linking photorespiration to NO3 assimilation in C3 plants - increased CO2 or decreased O2 levels (therefore more 'effective' Rubisco) leads to a decrease in nitrate assimilation, obviously as nitrogen is one of the most important macronutrients for crop plants reduced nitrogen assimilation is a bad thing for yield in crop plants - if photorespiration is somehow linked to nitrate assimilation then rubisco could be acting as a dual functional enzyme in C3 plants (in C4 plants the same effect was not seen, not that one would expect it to be as C4 rubisco exists in CO2 saturated environment and therefore works near optimal levels)

Anonymous said...

i've come across two reasons for why the oxygenase activity of Rubisco might be a good thing:

1. leads to fixed nitrogen
2. protects photosystems from light damage by recycling reducing cofactor NADH.

These are advantages for non-cultivated crops, but in controlled conditions could remove oxygenase activity and increase efficiency.

PS. How about engineering a rubisco that accepts a bicarbonate ion instead of CO2 (as PEP carboxylase does in C$ plants?