The evolution of citrate synthase

Citrate synthase [EC 2.3.3.1] is one of the key enzymes of the citric acid cycle. It catalyzes the joining of acetyl-CoA and oxaloacetate to produce citrate.

acetyl-CoA + H₂O + oxaloacetate → citrate + HS-CoA + H⁺

We usually think of this reaction in terms of energy production since acetyl-CoA is the end product of glycolysis and the citric acid cycle produces substrates that enter the electron transport system leading to production of ATP. However, it's important to keep in mind that the enzyme also catalyzes the reverse reaction.

There's another enzyme that catalyzes the same reaction using ADP/ATP as a cofactor. Citrate lyase [EC 2.3.3.8] is usually thought of as catalyzing the reverse reaction.¹

citrate + ATP + HS-CoA → oxaloacetate + acetyl-CoA + PO₄^2-

Citrate lyase is found in almost all species (unlike citrate synthase) and it catalyzes an important step in the reverse citric acid cycle where two molecules of CO₂ are fixed in the form of acetyl-CoA. Acetyl CoA is a "high-energy" compound used in the synthesis of fatty acids, which are essential components of lipids.

The structure of citrate synthase has been known for decades but citrate lysase is a "crystallization-recalitrant" enzyme, which is a fancy way of saying that lots of graduate students and post-docs have wasted a good deal of time trying to grow crystals without success. However, a group in Belgium recently succeeded in crystalizing the active domain of the enzyme by cutting out the region that was preventing crystallization (Verschueren et al., 2019).

As shown in Figure 4 of their paper (right), they noticed a remarkable similarity between the CoA binding domain of citrate lyase (CCL) and the active site of citrate synthase, which binds citate and CoA. This result leads to a very plausible scenario where citrate synthase evolved from a primitive citrate lyase.

The steps involve a duplication of the CoA-binding domain followed by a subsequent fusion event. The scheme is outlined in the paper (see below).

We will never be able to prove conclusively that this was the pathway leading to the evolution of a citrate synthase gene but that's not the point. The point is that this is a perfectly reasonable scenario based on the best available evidence from the structure and sequences of the two enzymes.² It's also consistent with our understanding of early life because the first bacteria probably used the citric cycle (Krebs cycle) reactions in order to fix CO₂, not to produce it. It's only much later on that bacteria acquired the ability to store sugars as glycogen that could then be broken down by glycolysis to produce acetyl-CoA in abundance. At that point, the ability to fix acetyl-CoA into citrate for subsequent oxidation in the citric acid cycle became important and the evolution of a citrate synthase became beneficial. (The figure below is also from the paper.)

If you've read this far, I'd like to indulge your patience for a few more minutes in order to make a point about teaching undergraduate biochemistry. The standard pathways that are found in the textbooks are extremely biased toward mammalian biochemistry. That's partly understandable because we are mammals and because many undergraduate courses are (inappropriately) focused on preparing students for medical school.

This explains why students see glycolysis as the most important pathway when, in fact, it's gluconeogenesis that's really important in most species. This explains why students see the citric acid cycle as a basic pathway when, in fact, many bacteria don't even use this pathway. This bias in teaching creates confusion in the minds of those students who go on to become researchers because they will have an incorrect view of the evolution of metabolic pathways. I think we should make an attempt to explain to students that humans are the end products of three billion years of evolution and they have adapted to a specialized way of life that relies on ingesting and metabolizing complex organic chemicals. The pathways that we see as "normal" in humans humans reflect this specialization. They are not the major pathways used in other species and they are not the most primitive pathways.

If we did this, then in future years we will never see papers claiming that glycolysis and the citric acid cycle are two of the most primitive pathways. That's a persistent misunderstanding of evolution that traces back to faulty teaching of undergraduates.

Background Reading

The evolution of the citric acid cycle
Fixing carbon by reversing the citric acid cycle
How not to teach biochemistry
Time to Re-Write the Textbooks! Nature Publishes a New Version of the Citric Acid Cycle

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1. The accepted name of this enzyme, according to IUBMB, is ATP citrate synthase.

2. This is an important point for creationists and intelligent design advocates who often argue that the evolution of genes such as the citrate synthase gene can never be explained by evolution.

Verschueren, K.H., Blanchet, C., Felix, J., Dansercoer, A., De Vos, D., Bloch, Y., Van Beeumen, J., Svergun, D., Gutsche, I., and Savvides, S.N. (2019) Structure of ATP citrate lyase and the origin of citrate synthase in the Krebs cycle. Nature, 568:571-575. [doi: 10.1038/s41586-019-1095-5]