Two simple molecules can react to form most of the intermediates in an analog of the modern-day Krebs cycle, without the need for enzymes or metals, according to a new study (Nat. Chem. 2020, DOI: 10.1038/s41557-020-00560-7). The findings give new insights into how this central metabolic cycle might have emerged, as well as potential commercial applications.
The Krebs cycle, also called the tricarboxylic acid (TCA) cycle, is the main way aerobic organisms release stored energy. In addition, the cycle produces precursors of some amino acids. Because it’s so fundamental to life on Earth, scientists are interested in studying how it might have emerged from prebiotic chemistry on early Earth. But working out this early chemistry is difficult. Typically, researchers trying to recreate possible prebiotic reactions in the lab focus on generating modern biomolecules, which might not be realistic without enzymes.
This focus on modern biomolecules hasn’t been fruitful because it has required harsh conditions and inorganic catalysts that can also break down the same building blocks, says Greg Springsteen, a chemist at Furman University, who led the new study along with Ramanarayanan Krishnamurthy, a chemist at Scripps Research. Both researchers are members of the Center for Chemical Evolution (CCE).
In previous work, Krishnamurthy, Springsteen, and coworkers had shown that they could generate two intermediates in the Krebs cycle by mixing pyruvate, glyoxylate, and hydrogen peroxide. Extending that work, they focused instead on α-ketoacids, a class of biomolecules that are related to but more reactive than the carboxylates formed in the Krebs cycle. They thought this extra reactivity would allow them to avoid harsh conditions and metal catalysts. Mixing pyruvate and glyoxylate in phosphate buffer at pH 7 resulted in the formation of five α-ketoacids, all of which are analogs of intermediates in the Krebs cycle.
“Pyruvate is the carbon skeleton, and then everything else is done by glyoxylate,” Springsteen says. “These are the kinds of reactions that make people throw in inorganic catalysts and harsh conditions because they wouldn’t suppose these reactions would go so well under mild conditions.”
The glyoxylate even acted as a reductant in one reaction. “That was a complete surprise,” Krishnamurthy says.
Last year, Joseph Moran of the University of Strasbourg and coworkers reported that they could recapitulate the Krebs cycle by mixing pyruvate and glyoxylate in the presence of ferrous ion (Nature 2019, DOI: 10.1038/s41586-019-1151-1). While Krishnamurthy thinks the iron isn’t needed for the synthesis of Krebs cycle intermediates, Moran’s work suggests it is needed for the breakdown. “Life builds up molecules and breaks them down in a steady state, just as a fountain pumps water up and lets it fall back down under gravity,” Moran writes in an email.
The new study is “a wonderfully interesting and instructive piece of work that demonstrates experimentally an alternative path for how the Krebs cycle might have emerged in biochemistry,” Paul Bracher, who studies prebiotic chemistry at Saint Louis University and is also a member of the CCE but was not involved in the study, says via email. He adds that when run in reverse, the Krebs cycle “could have been responsible for building organic molecules from carbon dioxide and water.”
Nicholas V. Hud, a chemist at the Georgia Institute of Technology who is a colleague of the study’s authors, says the work shows that reactions analogous to those in the Krebs cycle can happen without the need for catalysts. This supports the idea that “organic molecules with the right balance of reactivity and stability, like glyoxylate, were able to ‘get the ball rolling’ towards life,” he writes in an email. Those organic molecules, he believes, were in time replaced by functionally superior molecules that required the emergence of enzymes to be formed.
The chemistry could also have modern-day applications. Springsteen and his student R. Trent Stubbs have started a company, Aconabolics, to harness the chemistry for making isotopically labeled metabolites for medical diagnostics without the need for enzymes or fermentation. “To convert from the analogs that we make in this pathway to the modern biomolecules is an easy reaction,” Springsteen says.