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Biochemisty and the assembly of ammonium machines

Researchers who intend to build enzymes in the laboratory need detailed knowledge about the function of the respective enzymes. Using modern biochemistry methods, researchers led by Prof. Dr. Oliver Einsle from the University of Freiburg have in the last few years clarified the atomic structure of a complex bacterial protein that converts atmospheric nitrogen into a form that can be readily used by other organisms. The researchers have recently been awarded a European Research Council (ERC) Starting Grant, which they will now use to stabilize and optimize this enzyme complex in model organisms instead of in their natural hosts. If they succeed, they might at some time in the future be able to replace synthetic fertilizer.

Nitrogen is an essential component of amino acids and hence of all proteins. However, plants are unable to directly use atmospheric gaseous nitrogen as it is present as almost inert nitrogen (N2). Synthetic fertilizer must therefore be applied to fields where potatoes, soy and maize are grown in order to provide the crops with nitrogen in the form of nitrate salts. Plants supplied with adequate nitrogen grow rapidly and produce large amounts of green foliage, seeds and fruit. Poorer countries in Africa, for example, cannot afford to buy the expensive fertilizer whereas richer European countries, for example, usually apply too much fertilizer, which leads to environmental pollution. “The question is whether it might be possible to manipulate plants in such a way as to make them able to convert atmospheric nitrogen into a biologically usable form themselves so that they will cease to be dependent on the application of synthetic fertilizer,” said Prof. Dr. Oliver Einsle from the Institute for Organic Chemistry and Biochemistry at the University of Freiburg. “This might be possible if we had in-depth knowledge about the biochemistry of bacteria that are part of the atmospheric nitrogen cycle.”

Towards the synthesis of artificial enzymes

Model consisting of iron and sulphur atoms and a molybdenum atom in the active centre of the nitrogenase enzyme. © Prof. Dr. Oliver Einsle

Nitrogen occurs in the Earth’s atmosphere either as gaseous nitrogen (N2) or in oxidized form (NO2, N2O and NO). This nitrogen is not directly available to plants, but is converted into available forms by microorganisms. Bacteria convert gaseous nitrogen into ammonium ions (NH4+) in a biochemical process known as nitrogen fixation. The task is accomplished through an enzyme called nitrogenase. Over the last few years, Einsle and his team have refined the existing conceptions about the structure of this enzyme using biochemical methods such as crystal structure analysis. They have also looked into the active centre of the enzyme and shown that the decisive reaction of nitrogen fixation is mediated by the largest known structure consisting of iron, molybdenum and sulphur atoms. They now have detailed knowledge about this reaction and will apply this knowledge to the modification of plant cells. 

The researchers will use a synthetic biology approach. Einsle and his colleagues were awarded a prestigious European Research Council (ERC) Starting Grant for their N-ABLE project in July 2012. The researchers will use the €1.64 million award to transfer their knowledge to an artificial system. They plan to use simple E. coli cells and equip them with the machinery necessary for fixing nitrogen. But this is far from being a trivial challenge, as the enzyme nitrogenase consists of three subunits. In addition, at least twenty genes are involved in the synthesis of the enzyme. Some gene products are responsible for the correct folding of the individual components and their correct assembly, others deliver the necessary energy by cleaving ATP and pumping electrons across the membranes in order to build up electrochemical potentials.

A wide variety of interests and clinical perspectives

“We plan to use biochemical methods to identify all the individual mechanisms required to establish the system,” said Einsle, going on to add, “and in parallel, we plan to put together the individual components step by step in E. coli and investigate which components need to come together when and in what order for the decisive reaction to occur.” The researchers plan to develop a kind of genetic module that can theoretically also be integrated into a plant cell, or in other words, they hope to develop a nitrogenase machinery that fits in naturally with the plants’ daily business and produces ammonium ions from atmospheric nitrogen. However, these are only pipe dreams. Einsle’s group of researchers has a wide variety of interests, and they are also interested in what the project will teach them about the biochemical mechanisms involved.

This protein complex, which consists of five subunits, transports the salt of formic acid and is located in the membrane of bacterial cells. © University of Freiburg

In their research into the enzymes involved in the bacterial nitrogen metabolism, Einsle’s team of researchers also discovered other enzyme complexes. One of these is a complex located in the membrane of bacterial cells, which consists of five different protein subunits and transports formic acid salt across the bacterial cell membrane. Formic acid is one of the metabolic products of bacteria found in the human intestines. It can be used by specialized microorganisms – including pathogens such as Salmonella – as an additional source of energy. A recent paper published by Einsle’s group reports on their success in clarifying the structure of the protein complex and obtaining important information about the transport mechanisms. “I am sure that this knowledge will one day help us develop drugs to target the formic acid salt transport mechanisms and weaken pathogens like Salmonella,” said Einsle. 

The participation of Einsle’s team in the cooperative research centre “Medical epigenetics – from basic mechanisms to clinical applications” (SFB 992) is also of clinical relevance. This project is focused on the identification of small molecule inhibitors of epigenetic proteins with the goal to modify the programming of cancer cells, to name but one example. “All our projects have application-oriented perspectives,” said Einsle. “But research still needs to do some biochemistry homework before it is able to turn the knowledge gained into concrete products.”

Further information:
Prof. Dr. Oliver Einsle
Department of Biochemistry
Institute for Organic Chemistry and Biochemistry
University of Freiburg
Albertstraße 21
79104 Freiburg
Tel.: +49 (761) 203 6058
Fax: +49 (761) 203 6161
E-mail: einsle(at)bio.chemie.uni-freiburg.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/biochemisty-and-the-assembly-of-ammonium-machines