Chemists explore the mechanisms behind food spoilage

The research team of Annette Rompel from the Institute for Biophysical
Chemistry, University of Vienna explore the mechanisms behind the
"browning reaction" during the spoilage of mushrooms. The researchers
were able to demonstrate that the enzyme responsible is already formed prior to
fungal spoiling. The detailed study is published online in the well renowned
journals, Phytochemistry and Acta Crystallographica.

Understanding the mechanism of enzyme tyrosinase pigmentation is
currently of both medical as well as technological interest. The copper-containing
enzyme is present in animals and humans and is essential for the protective
pathway against UV radiation, it also simultaneously provides the elucidation
and potential means with which to prevent the spoilage of food. Mushrooms were
selected for the study due to their low cost and ready availability. In
addition they are also a valuable target for researchers, largely because of
their high enzyme content, fungi are ideal sources for potential studies of
tyrosinase. The mushrooms therefore serve as a model organism for the study of
the pathway involved in food spoilage.

Elucidation of an inactive
precursor

Since 2012, it has become established that six different tyrosinases
(PPO1 to 6) exist within the mushroom, two of which (PPO3 and PPO4) occur in
larger quantities. The enzyme responsible for the mechanism of food spoilage is
formed within eukaryotes (organisms that have a nucleus) as an inactive
precursor during the developmental phase of an organism. This precursor is then
activated via specific chemical cleavage. At this cleavage site, the protein
segment covering part of the enzyme active site is removed and the substrates
(tyrosine and other monophenols) can be accessed and take part in key chemical
reactions.

New Method of Isolation and
unique reagent leads to success

None of the previously established methods of isolation present in the
literature could be successfully applied for PPO4. For the first time, at the
Institute for Biophysical Chemistry of the University of Vienna, a method was
developed that allows for the one stage isolation of latent tyrosinase from
their natural source. The enzyme characterization was undertaken at the
Department of Chemistry with close cooperation with the Institute of Mass
Spectrometry lead by Andreas Rizzi, both within the University of Vienna. After
sufficiently large quantities were extracted from pure PPO4, the researchers
were able to identify and optimize appropriate crystallization conditions which
produced well formed protein crystals. This was only possible with the use of a
relatively unusual co-crystallization reagent, a polyoxometalate of the
Anderson type.

Integral for medicine and
biotechnology

The PhD student Stephan Mauracher worked at the University of Vienna,
taking an essential part in this project as part of the University Initiative
termed "Functional Molecules". Following this research it has been
made possible to purify the enzyme in sufficient quantities for
characterization. Ulrich Kortz, working at the Jacobs University of Bremen was
responsible for synthesizing the polyoxometalate used and was a key proponent
of its usage as an additive for protein crystallization.

The research project was then continued as FWF Initiative. "This
research has allowed for the crystallization and three-dimensional structure of
PPO4 to be resolved", says Anette Rompel.

Scientific contact

Univ.-Prof. Dr. Annette Rompel

Institute of Biophysical Chemistry

Chemistry Faculty

University of Vienna

T +43-1-4277-525 02

annette.rompel@univie.ac.at

http://www.bpc.univie.ac.at

The University of Vienna, founded in 1365, is one of the oldest and
largest universities in Europe.

IMAGE: The crystal structure of latent PPO4 mushroom tyrosinase is
converted via proteolytic removal of the C-terminal domain resulting in the
active form of tyrosinase. As a result of this reaction