"Magical" enzymes

Producing hydrogen efficiently with the help of hydrogenases while consuming electricity, or - conversely - generating electricity from hydrogen, is a dream of biotechnology. Researchers at the Max Planck Institute for Terrestrial Microbiology have now elucidated key metabolic steps in the biosynthesis of the [Fe]-hydrogenase cofactor. Their findings are not only an important step towards the in vitro biosynthesis of the hydrogenase itself, but also provide insight into the mode of action of a äußfirst versatile class of enzymes involved in this process.

Hydrogen gas (H2) is a versatile energy carrier and an important option for storing renewable energy. However, both the industrial production of hydrogen and fuel cells that use hydrogen require the rare and therefore expensive precious metal platinum.

Nature has found another solution. Since the beginning of the earth's history, microorganisms have utilised enzymes, in this case so-called hydrogenases. The enzymatic conversion of hydrogen takes place efficiently, with almost no energy loss and, above all, without greenhouse gas emissions. Two hydrogenases, the [NiFe]-hydrogenase and the [FeFe]-hydrogenase, have already been studied in detail worldwide. The third known hydrogenase, [Fe]-hydrogenase, has been discovered and structurally described in detail by researchers at the Max Planck Institute for Terrestrial Microbiology in Marburg. In an earlier study, the team led by Seigo Shima succeeded in recreating the central metallocofactor of the enzyme [Fe]-hydrogenase in a test tube.

Identification of two enzymes

In order to establish a defined system for the production of this enzyme in vitro, the biosynthesis chain must be fully clarified. In collaboration with EPFL Lausanne and the University of Minnesota, the team has now identified two important enzymes - the first and the last step - for the biosynthesis of the key element, the metallocofactor of [Fe]-hydrogenase.

Fascinatingly, both enzymes belong to the superfamily of "radical S-adenosyl-methionine (SAM) enzymes", which mediate a remarkable variety of radical-based reactions with substrates, from small organic molecules to proteins, DNA or RNA. Their versatility makes them promising catalysts for biotechnological applications, but also difficult candidates for the identification of substrates and mechanisms - both basic prerequisites for the use of enzymes as biochemical tools.

„Magical“ enzymes

The fact that radical SAM enzymes seem to operate "magically" is a real challenge for research, says Francisco Arriaza Gallardo, first author of the study: "Radical SAM enzymes are very creative enzymes. They can literally take two substrates apart and turn them into something completely new. This means that you cannot determine the substrate, even if you have identified the result."

To overcome these challenges, the scientists used their recently developed in vitro biosynthesis method, in which they investigated the enzymes with chemically synthesised precursors as building blocks. This new combination of synthetic precursors and biological materials made it possible for the first time to replicate the natural biosynthesis process outside of a living cell. By systematically combining the presumed parts of the reaction chain, the researchers were finally able to predict a function for each enzyme involved in [Fe]-hydrogenase production.

Research group leader Seigo Shima explains: "The next question is: Can we produce an active [Fe]-hydrogenase in a defined system without cellular components? Because only with the fully defined system can we identify all the components for the biosynthesis of this cofactor." Until then, some fine-tuning is still needed. Co-author Sebastian Schaupp recalls: "The idea for our system was the result of intensive brainstorming. We thought about what biosynthesis might need and put it together from the bottom up. Now we have to find out what can be left out."

By elucidating the catalytic mechanism, the team also hopes to gain insights into the design and biosynthesis of new catalysts in general. In addition, the confirmation of the crystal structures could lead to a better understanding of the "magic" radical SAM enzymes.

News of the Max Planck Society from 17 January 2023