Key enzyme in citrate cycle also functions “backwards”

The citrate cycle: most people will probably remember this metabolic pathway from biology lessons at school. It provides numerous organisms with energy by breaking down organic substances, thereby enabling them to live. Organisms ranging all the way from bacteria to humans use this pathway, consuming oxygen in the process.

Some microorganisms even use the citrate cycle when oxygen is absent – in other words, under anaerobic conditions – in the opposite direction: They build up biomass from carbon dioxide by means of the “reductive” citrate cycle. This means that they fix inorganic carbon, just like plants do in photosynthesis.

A team of researchers from the University of Münster, headed by biologist Prof. Ivan Berg, has unexpectedly discovered that a central enzyme in the citrate cycle functions both “forward” and “backwards”. Citrate synthase catalyzes the first step in the citrate cycle – the formation of citrate – thereby giving it its name. It had previously been regarded impossible that this step could be reversed in living cells – but not any longer.

The researchers have discovered that in certain anaerobic bacteria, which are able to fix inorganic carbon by means of the reductive citrate cycle, citrate synthase functions backwards and cleaves citrate instead of forming it. The researchers’ findings have been published in the latest issue of the journal “Science”.

“Shortly before our discovery I was still telling students in my lectures that under physiological conditions – in other words, in living cells – the citrate synthase reaction can only take place in one direction,” recalls Ivan Berg from the Institute of Molecular Microbiology and Biotechnology at Münster University. “Our latest findings disprove a decade-old conventional wisdom.” In addition to the Münster team, other researchers were also involved in the study – from the University of Freiburg, the Technical University of Munich (co-corresponding author: Prof. Wolfgang Eisenreich) and the Water Technology Centre in Karlsruhe.

From an energetic point of view, the newly discovered variation in the reductive citrate cycle is the most efficient way to fix carbon. In contrast to the variations previously known, no energy is used in the form of the energy carrier adenosine triphosphate (ATP) to cleave the citrate into acetyl coenzyme A and oxaloacetate. The enzyme, which normally enables citrate to be cleaved in the reductive citrate cycle, is a so-called ATP-dependent citrate lyase. This enzyme was seen as the key enzyme of the “reverse” pathway.

Experts describe organisms which feed on organic compounds as “heterotrophic”, in contrast to organisms which build up biomass with the aid of light energy or chemical energy, which are called “autotrophic”. The results of this new study have implications for our understanding of their evolution.

“With the possibility of using the enzymes of the heterotrophic oxidative citrate cycle for autotrophic metabolism, an autotrophic organism can develop very easily from a heterotrophic one,” says Ivan Berg. “Our results indicate that, in the evolutionary process, the capability to grow autotrophically has developed independently on several occasions.” In the researchers’ view, many anaerobic microorganisms are potential autotrophs.

The researchers’ discovery arose by chance. “While we were looking for the autotrophic carbon fixation pathway in a bacterium called Desulfurella acetivorans, we unexpectedly observed ATP-independent citrate cleavage,” says lead author Achim Mall, a member of Ivan Berg’s team. Using nuclear magnetic resonance spectroscopy, the researchers demonstrated at a molecular level that citrate synthase makes this reaction possible.

“Our results show that unexpected discoveries are possible even when investigating thoroughly investigated metabolic pathways,” says Ivan Berg. “There are probably further surprises awaiting us in the field of metabolic biochemistry.”

Desulfurella acetivorans was discovered on the north-east Asian Kamchatka Peninsula in the late 1980s in a volcanic spring with a temperature of over 50°C. The bacterium is strictly anaerobic and respires elemental sulfur instead of oxygen. It can live either heterotrophically, feeding on acetate or other organic substrates, or autotrophically, fixing inorganic carbon and gaining energy from the oxidation of hydrogen.

The research work received funding from the German Research Foundation and the Hans Fischer Society, Munich.

Original publication:

Mall A. et al. (2018): Science 02 Feb 2018: Vol. 359, Issue 6375, pp. 563-567; DOI: 10.1126/science.aao2410

https://www.uni-muenster.de/Biologie.IMMB/en/Arbeitskreise/agberg/index.html Group Prof. Dr. Ivan Berg