Scientists provide new insights into the citric acid cycle

(Press-News.org) The citric acid cycle is an important metabolic pathway that enables living organisms to generate energy by degrading organic compounds into carbon dioxide (CO?). The first step in the cycle is usually performed by the enzyme citrate synthase, which builds citrate. But, in the absence of oxygen (under anaerobic conditions), some bacteria can perform the reverse cycle: They can build up biomass from CO?. In this so-called reversed citric acid cycle, citrate synthase is replaced by ATP-citrate lyase, which consumes cells' universal energy carrier ATP (adenosine triphosphate) to cleave citrate instead of forming it. However, a few years ago, a research team led by Ivan Berg (University of Münster) and Wolfgang Eisenreich (Technical University of Munich) discovered that instead of requiring ATP-citrate lyase for the reversed cycle, some anaerobic bacteria can use citrate synthase itself to catalyze citrate cleavage without consuming ATP. Now, the same team found that bacteria using this metabolic pathway (the reversed citric acid cycle through citrate synthase), depend on very high concentrations of both the enzyme and carbon dioxide.

As a comparison, the CO? concentration in air is around 0.04%, but bacteria using this pathway require at least 100 times more than that for their growth. The researchers assume that such CO? concentration-dependent pathways could have been widespread on the primordial earth, since the CO? concentration was high at the time. Therefore, this metabolic pathway may be a relic of early life. The results of the study have been published in the journal Nature (online in advance).

The team studied the anaerobic bacteria Hippea maritima and Desulfurella acetivorans. These organisms live without oxygen in hot springs, where the CO? concentration can be 90% and higher. "It is conceivable that many other organisms use this cycle to bind CO?," says Ivan Berg. "Our findings are in line with the results of several recent studies highlighting a potential widespread occurrence of this reversed oxidative citric acid cycle." Nevertheless, many bacteria use the energetically less efficient ATP-dependent reaction for citrate cleavage. "It was mysterious why this 'expensive' version of the pathway exists if an energetically much cheaper alternative through the backwards reaction of citrate synthase is feasible. Now we know that this is due to the low CO? concentrations in many environments. The cheap alternative doesn't work there." emphasizes Wolfgang Eisenreich.

These findings could also be of interest for biotechnology. With the knowledge that autotrophic organisms using this "backward cycle" depend on the CO? concentration, scientists can apply it to more efficiently convert substrates into value-added products.

The results in detail

The scientists wanted to understand what factor determines whether the citric acid cycle runs "forwards" or "backwards" in the bacteria. Cultivating the bacteria under different conditions, they noticed that the growth of these organisms was highly dependent on the CO? concentration in the gas phase. In detail, the high CO? concentration was needed to allow the function of another important enzyme, pyruvate synthase. This enzyme is responsible for assembling acetyl coenzyme A (acetyl-CoA), the product of the "reversed cycle". The high CO? concentration drives the pyruvate synthase reaction in the direction of carboxylation and the entire cycle backwards, enabling CO? to be converted into biomass. The studied Hippea maritima and Desulfurella acetivorans were able to grow very well at 20% and 40% CO? in the gas phase but only moderately at 5% CO?, and no growth was possible at 2% or 1% CO?. As a control, the scientists studied another autotrophic bacterium Desulfobacter hydrogenophilus, which uses the energetically more expensive ATP-citrate lyase version of the reversed citric acid cycle. In this bacterium, growth was not affected by the CO? concentration.

The "backward cycle" that uses citrate synthase for citrate cleavage cannot be bioinformatically predicted, as it does not have the key enzymes whose presence can be used as a marker for the functioning of the pathway. Therefore, as an identifying feature for bioinformatic analyzes, the scientists used the detected high levels of citrate synthase in these bacteria's protein cocktail. Using a special analysis tool, the researchers were able to predict the production levels of individual proteins. With this trick, it was possible to predict the functioning of the "backward cycle" for inorganic carbon fixation in many anaerobic bacteria.

The scientists also showed that no gene regulation was necessary for switching from the oxidative ("forward") to the reductive ("reverse") direction. "This means that the cells can react very quickly on the availability of the carbon source in the environment" says Ivan Berg. "They use either the reductive direction to fix CO?, if the concentration of CO? is high, or the oxidative direction, if another carbon source is available."

Methodological notes

The methods used in the study were mass spectrometry and 13C-isotope analyses, enzyme measurements, protein quantification as well as media and amino acid analyses using chromatographic and spectrometric methods (LC/MS or GC/MS). With bioinformatic methods, they examined the occurrence of certain nucleotide base combinations (codons) in order to make predictions about the production of individual proteins.

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