Researchers found missing links for the in vitro biosynthesis of [Fe]-hydrogenase
Using hydrogenases to efficiently produce hydrogen with electricity or generate electricity from hydrogen would make a biotechnology dream come true. Now researchers from the Max Planck Institute for Terrestrial Microbiology have clarified crucial catalytic steps in the biosynthesis of the [Fe]-hydrogenase cofactor. Their findings not only pave the way towards in vitro biosynthesis of the hydrogenase itself, but also give insights into the enzymatic action of highly versatile enzymes that are part of the process.
Hydrogen gas (H2) is a versatile energy carrier and one of the leading options for storing renewable energy. However, industrial production of hydrogen and fuel cells that utilize hydrogen need the rare and thus expensive precious metal platinum.
Nature, on the other hand, has found a different solution. Since earth`s ancient times, microorganisms use enzymes, so-called hydrogenases. Enzymatic conversion of hydrogen is efficient, almost without energy loss, and, most importantly, without greenhouse gas emissions. Two hydrogenases, [NiFe]-hydrogenase and [FeFe]-hydrogenases, have been studied extensively worldwide. The third known hydrogenase, [Fe]-hydrogenase, was discovered and structurally described in detail at the Max Planck Institute for Terrestrial Microbiology in Marburg. In a previous work, scientists led by Seigo Shima were able to recreate the central metallocofactor of the enzyme [Fe]-hydrogenase in a test tube.
In order to establish a defined system for the production of this enzyme in vitro, the basic principles of biosynthesis – the biosynthetic chain - had yet to be fully resolved. In collaboration with the EPFL Lausanne and University of Minnesota, the team was now able to identify two important enzymes – the first and the last step - for the biosynthesis of the key element, the [Fe]-hydrogenase metallocofactor.
Fascinatingly, these enzymes belong to the superfamily called “radical S-adenosyl methionine (SAM) enzymes”, which catalyze remarkably diverse variety of radical-based reactions on substrates ranging from small organic molecules to proteins, DNA or RNA. Their versatility makes them very promising catalysts for biotechnological applications, but at the same time tough candidates for identifying substrates and mechanism – both a prerequisite for using these enzymes as biochemical tools.
According to Francisco Arriaza Gallardo, main author of the study, the fact that radical SAM enzymes seem to operate quite “magically” is a true challenge in terms of research: “Radical SAM enzymes are very creative enzymes. They can literally take two substrates apart and make totally new things. This means that you cannot estimate the substrate even when you have identified the outcome.”
Probing the enzymes with chemically synthesized precursors
In order to tackle these challenges, the scientists used their recently developed in vitro biosynthesis method, probing the enzymes with chemically synthesized precursors as building blocks. This new combination of synthetic precursors and biological materials enabled, for the first time, to replicate the natural biosynthesis process outside of a living cell. Combining the assumed parts of the reaction chain in a systematic manner, they were finally able to predict a function for every enzyme of the [Fe]-hydrogenase production. Research group leader Seigo Shima pointed out: “The next question is: can we produce an active [Fe]-hydrogenase in a defined system without cellular components? This is because we can identify all components for biosynthesis of this cofactor only by using the fully defined system”.
For this, some fine-tuning will be necessary. Co-author Sebastian Schaupp recalls: “The idea for our system was the result of an intense brainstorming. We proposed what the biosynthesis might need and put it together from the bottom up. Now it is important to find out what can be left out.” By identifying the catalytic mechanism, the team wants to gain insight into the design and biosynthesis of new catalysts in general. In addition, confirming the crystal structures might led to a better understanding of the “magical” radical SAM enzymes.