AauDyP, a DyP peroxidase enzyme found in tree ear mushrooms, and other members of the haem peroxidase family are the major field of research of Prof. Dr. Dietmar Plattner’s research group at the Institute of Organic Chemistry at the University of Freiburg. Plattner’s co-workers, Dr. Klaus Piontek and Eric Strittmatter along with colleagues from the International Institute Zittau, have clarified the atomic structure of AauDyP using crystallographic methods. Their objective is to obtain in-depth insights into peroxidase function in order to be able to produce enzymes that can be used in biotechnology.
Tree ears (Auricularia auricula-judae), which grow primarily on the wood of old elderberry bushes and are shaped like an ear, contain numerous enzymes that help the mushrooms secure their nutrition. Among these enzymes are peroxidases that catalyse the reduction of hydrogen peroxide (compounds containing reactive oxygen-oxygen bonds) and oxidise various substrates. Peroxidase enzymes are particularly common in fungi and bacteria, but are also found in humans where they are involved in the steroid and fatty acid metabolisms. Peroxidases also eliminate toxins that would otherwise be damaging to the body. “This is, however, only a desirable side effect which can be used for biotechnological applications,” said Prof. Dr. Dietmar Plattner from the Institute of Organic Chemistry at the University of Freiburg, going on to add, “but it is not their primary biological purpose.” Such fungal enzymes can, amongst other things, be of interest to humans due to their ability to degrade lignin, a component of wood that can only be degraded by a handful of organisms. They can also be used to decolourise dyes, and have become popular agents for stonewashing denim.
For many years, Plattner’s research group has been focusing on haem peroxidases, enzymes that contain haem as central co-factor. Haem constitutes the deep red pigment portion of haemoglobin. Tree ear DyP (dye decolourising) peroxidase is one of many haem peroxidases. Plattner’s group of researchers has recently succeeded in elucidating its structure, a challenge that was far from simple. The researchers had to establish a pure fungus culture from which they were able to isolate the enzyme. They then had to grow a single, morphologically distinct crystal, a process that takes rather a long time to complete. The crystal’s regular, highly symmetrical structure can then be analysed in detail using X-ray crystallography, which is a relatively complex and time-consuming method. This can only be done at the ESRF (European Synchrotron Radiation Facility), which is why the team needed to travel to Grenoble in France to use the high irradiation intensity of the synchrotron for analysing matter on the atomic level. This method creates a defraction image of the enzyme crystal, which reveals the distribution of the electrons in the molecule.
“This image is visualised as a kind of coat or envelope around individual atoms,” said Dr. Klaus Piontek, explaining that the image only shows the outer wrapping from which the structure of the molecule can be reconstructed. Plattner compares the underlying principle with a microscope, where the light of a lens produces an image. “We do not have an X-ray lens,” said the chemist. “We use a computer to analyse our data, and this computer works rather like a lens that produces an image.”
Dietmar Plattner, Klaus Piontek, Eric Strittmatter and their colleagues from the International Institute Zittau have found out that two amino acids play a key role in the work of DyP peroxidases. “We have shown that a particular tyrosine on the surface of the enzyme is responsible for the interaction of the enzyme with the substrate,” Piontek explains. This tyrosine takes an electron from the substrate, thereby initiating the flow of electrons that eventually leads to the decolourisation of dyes. The electron is transferred to the central iron atom of the enzyme’s haem group by way of a second amino acid.
The team of researchers, consisting of chemists and structural biologists, has also been able to show using crystallographic methods that the DyP peroxidase under investigation has two different indentations (binding pockets) that can bind substrate molecules of different sizes. This enables the enzyme to convert two different compounds.
Why is it so interesting to explore molecules down to the last detail? “In addition to carrying out classical chemical syntheses, we are increasingly interested in learning from nature because we believe that this helps us transfer the function of the natural enzyme to chemical processes,” said Plattner explaining what biomimetic synthesis is all about. Detailed know-how on enzyme structure and reactions is of particular importance for molecules that either cannot be synthesised or can only be synthesised with difficulty.
As part of “green chemistry”, the use of enzymes adapted to industrial application conditions is an environmentally-friendly alternative to substituting harsh chemical reagents. Insights into peroxidase-catalysed processes also provide the researchers with the possibility of improving certain enzyme properties. “How can enzymes be modified in order to make them better suited for industrial application?” is one of the questions Piontek is interested in solving and specifically refers to the ability to either expand or limit the enzyme’s substrate spectrum. Eric Strittmatter, who is the only biologist in the team, knows the answer: “Genetic engineering methods can be used to produce enzyme variants by exchanging an amino acid in the enzyme’s binding pocket. The binding pocket can then bind different substrates. This is a good way of controlling the substrates an enzyme is able to metabolise.” In addition, such mutations also have the potential to maintain the activity of the enzyme in organic solvents such as acetone or alcohol, in which the enzyme would normally denature and become ineffective.
The team was part of the four-year EU-funded project 'BIORENEW: White biotechnology for added value products from renewable plant polymers: design of tailor-made biocatalysts and new industrial processes’ (2006 – 2010), and is now continuing its research as part of the ongoing BioIndustry 2021 programme. In view of the basic financing provided by the Baden-Württemberg government, the members of the team consider their research at the university a hobby. “If we all only worked a 40-hour week, results would be non-existent,” said one of the researchers involved in the project.
Further information:Prof. Dr. Dietmar A. Plattner / Klaus PiontekInstitute of Organic ChemistryAlbertstraße 2179104 FreiburgTel.: +49 (0)761/203-6013 or -6036E-mail: Dietmar.Plattner(at)chemie.uni-freiburg.deKlaus.Piontek(at)ocbc.uni-freiburg.de