On Not Getting with the Adaptationist ProgramAs 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.
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