I just realized that I don't have a post devoted to the evolution of the citric acid cycle. This need to be remedied since I often talk about it. It's a good example of how an apparently irreducibly complex pathway can arise by evolution. It's also a good example to get students to think outside of the box. Undergraduate biochemistry courses usually concentrate on human physiology and too often students transfer that bias to all other species. They assume that what happens in humans is what happens in plants, fungi, protozoa, and bacteria.1Here's what the standard citric acid cycle looks like (Moran et al., 2011 p. 393).
When the pathway runs in the clockwise direction, the net reaction is oxidation of a two-carbon compound (acetate, shown in green) to two molecules of CO2. The oxidation steps are coupled to reduction of NAD+ or Q and the production of an ATP equivalent at step 5. The substrate, acetyl-CoA, can be though of as a "high energy" molecule and the oxidation steps result in the release of energy that's captured in the synthesis of reducing equivalents (NADH2 and QH2). These reducing equivalents are used in multiple reactions inside the cell. They are usually thought of as the substrates for a series of membrane-associated electron transport reactions that create a proton gradient but that's only part of their fate. The proton gradient is used to drive ATP synthesis so you can think of the reducing equivalents as ATPs where NADH2 is equivalent to 2.5 ATPs and QH2 is equivalent to 1.5 ATPs.
Better BiochemistryIt's difficult to imagine how a cyclic pathway like this could evolve since in the absence of any one of the enzymes the circle is broken. You might naively think that all of the enzymes had to arise simultaneously.
In fact, we have a pretty good idea how the cyclic pathway evolved from more simple non-cyclic pathways. This is how I describe it in my textbook (Moran et al., 2011 pp. 412-414 © Pearson/Prentice Hall).
13.9 Evolution of the Citric Acid Cycle
The reactions of the citric acid cycle were first discovered in mammals and many of the key enzymes were purified from liver extracts. As we have seen, the citric acid cycle can be viewed as the end stage of glycolysis because it results in the oxidation of acetyl CoA produced as one of the products of glycolysis. However, there are many organisms that do not encounter glucose as a major carbon source and the production of ATP equivalents via glycolysis and the citric acid cycle is not an important source of metabolic energy in such species.
We need to examine the function of the citric acid cycle enzymes in bacteria in order to understand their role in simple single-celled organisms. These roles might allow us to deduce the pathways that could have existed in the primitive cells that eventually gave rise to complex eukaryotes. Fortunately, the sequences of several hundred prokaryotic genomes are now available as a result of the huge technological advances in recombinant DNA technology and DNA sequencing methods.We can now examine the complete complement of metabolic enzymes in many diverse species of bacteria and ask whether they possess the pathways that we have discussed in this chapter. These analyses are greatly aided by developments in the fields of comparative genomics, molecular evolution, and bioinformatics.
Most species of bacteria do not have a complete citric acid cycle. The most common versions of an incomplete cycle include part of the left-hand side. This short linear pathway leads to production of succinate or succinyl CoA or α-ketoglutarate by a reductive process using oxaloacetate as a starting point. This reductive pathway is the reverse of the traditional cycle that functions in the mitochondria of eukaryotes. In addition, many species of bacteria also have enzymes from part of the right-hand side of the citric acid cycle, especially citrate synthase and aconitase. This allows them to synthesize citrate and isocitrate from oxaloacetate and acetyl CoA. The presence of a forked pathway (Figure 13.24) results in the synthesis of all the precursors of amino acids, porphyrins, and fatty acids.
There are hundreds of diverse species of bacteria that can survive and grow in the complete absence of oxygen. Some of these species are obligate anaerobes—for them, oxygen is a lethal poison! Others are facultative anaerobes—they can survive in oxygen free environments as well as oxygen-rich environments. E. coli is one example of a species that can survive in both types of environment. When growing anaerobically, E. coli uses a forked version of the pathway to produce the necessary metabolic precursors and avoid the accumulation of reducing equivalents that cannot be reoxidized by the oxygen requiring electron transport system. Bacteria such as E. coli can grow in environments where acetate is the only source of organic carbon. In this case, they employ the glyoxylate pathway to convert acetate to malate and oxaloacetate for glucose synthesis.
The first living cells arose in an oxygen-free environment over three billion years ago. These primitive cells undoubtedly possessed most of the enzymes that interconverted acetate, pyruvate, citrate, and oxaloacetate, since these enzymes are present in most modern bacteria. The development of the main branches of the forked pathway possibly began with the evolution of malate dehydrogenase from a duplication of the lactate dehydrogenase gene. Aconitase and isocitrate dehydrogenase evolved from enzymes that are used in the synthesis of leucine (isopropylmalate dehydratase and isopropylmalate dehydrogenase, respectively). (Note that the leucine biosynthesis pathway is more ubiquitous and more primitive than the citric acid cycle.)
Extension of the reductive branch continued with the evolution of fumarase from aspartase. Aspartase is a common bacterial enzyme that synthesizes fumarate from L-aspartate. L-aspartate, in turn, is synthesized by amination of oxaloacetate in a reaction catalyzed by aspartate transaminase (Section 17.3). It is likely that primitive cells used the pathway oxaloacetate → aspartate → fumarate to produce fumarate before the evolution of malate dehydrogenase and fumarase. The reduction of fumarate to succinate is catalyzed by fumarate reductase in many bacteria. The evolutionary origin of this complex enzyme is highly speculative but at least one of the subunits is related to another enzyme of amino acid metabolism. Succinate dehydrogenase, the enzyme that preferentially catalyzes the reverse reaction in the citric acid cycle, is likely to have evolved later on from fumarate reductase via a gene duplication event.
The synthesis of α-ketoglutarate can occur in either branch of the forked pathway. The reductive branch uses α-ketoglutarate:ferredoxin oxidoreductase, an enzyme found in many species of bacteria that don’t have a complete citric acid cycle. The reaction catalyzed by this enzyme is not readily reversible. With the evolution of α-ketoglutarate dehydrogenase the two forks can be joined to create a cyclic pathway. It is clear that α-ketoglutarate dehydrogenase and pyruvate dehydrogenase share a common ancestor and it is likely that this was the last enzyme to evolve.
Some bacteria have a complete citric acid cycle but it is used in the reductive direction to fix CO2 in order to build more complex organic molecules. This could have been one of the selective pressures leading to a complete pathway. The cycle requires a terminal electron acceptor to oxidize NADH and QH2 when it operates in the more normal oxidative direction seen in eukaryotes. Originally, this terminal electron acceptor was sulfur or various sulfates, and these reactions still occur in many anaerobic bacterial species. Oxygen levels began to rise about 2.5 billion years ago with the evolution of photosynthesis reactions in cyanobacteria. Some bacteria, notably proteobacteria, exploited the availability of oxygen when the membrane associated electron transport reactions evolved. One species of proteobacteria entered into a symbiotic relationship with a primitive eukaryotic cell about two billion years ago. This led to the evolution of mitochondria and the modern versions of the citric acid cycle and electron transport in eukaryotes. The evolution of the citric acid cycle pathway involved several of the pathway evolution mechanisms discussed in Chapter 10. There is evidence for gene duplication, pathway extension, retro-evolution, pathway reversal, and enzyme theft.
Additional information on other enzymes in this pathway can be found in: On the Evolution of New Enzymes: Completely Different Enzymes Can Catalyze Similar Reactions.
For more information on succinate dehydrogenase and it's proper substrates and products see: Succinate Dehydrogenase; Succcinate Dehydrogenase and Evolution by Accident.
For more details on the relationship of &appha;-ketoglutarate dehydrogenase and pyruvate dehydrogenase see: Pyruvate Dehydrogenase Evolution.
1. The emphasis on human biochemistry and physiology—partly in order to prepare students for medical school—is what inhibits teaching evolutionary concepts in biochemistry courses.
Moran, L.A., Horton, H.R., Scrimgeour, K.G., and Perry, M.D. (2012) Principles of Biochemistry 5th ed., Pearson Education Inc.