19.8. Structural Studies of Ascomycete Laccases – Insights into the Reaction Pathways


 Public examination of a doctoral dissertation in the field of organic chemistry

Doctoral candidate: M.Sc. Juha Kallio 

Date and venue: 19.8.2011 at 12 noon, F100, Joensuu campus 


Laccases are a sub-group of copper-containing enzymes called multicopper oxidases. Laccases have an ability to oxidize both phenolic and non-phenolic lignin-related compounds as well as highly detrimental environmental pollutants. They use dioxygen as a terminal electron acceptor in a catalytic reaction, in which the oxygen is directly reduced to water molecules without producing harmful by-products. Some laccases and other multicopper oxidases also have a high electrochemical potential towards substrate oxidation. In addition, physicochemical properties, such as a wide range of optimal pH and high thermostability of some laccases enhance the potential of the laccase enzymes in industrial use. The biocatalytic properties of these enzymes have already been found to be useful in several biotechnological applications such as denim bleaching, as cleaning agents for water purification systems and as catalysts in organic synthesis. 

The properties and structures of laccases have long been examined for example by spectroscopic and electrochemical measurements, theoretical calculations and X-ray crystallography to determine the mechanism of the reaction catalyzed by these enzymes. However, the three-dimensional structure of a plant lacccase, the details of the dioxygen reduction and a possible role of carbohydrates is still unknown. Different laccases and other multicopper oxidases may also have adapted different approaches for their function by convergent evolution. These are the reasons why more effort in the research on laccases is still needed. Specific structural studies can give invaluable information for the relationship between the structure and function of enzymes. In the best cases, the results obtained can be used to design more efficient catalysts for applications through genetic modifications of the original enzymes. 

In this study, X-ray crystallography was used to determine the crystal structures of laccases from ascomycete fungi Melanocarpus albomyces and Thielavia arenaria. The crystal structures of M. albomyces laccase, both natively and heterologously expressed, were known and published before the beginning of this project and these structures were the only known crystal structures for laccases from ascomycete fungi. The structures of genetically modified M. albomyces laccase presented here are focused on specific aspects within the catalytic process: oxidation of phenolic substrates, role of the C-terminus and dioxygen entry. 

Six new crystal structures were released during this study, including the novel structure of T. arenaria laccase. We especially obtained insights into a buried C-terminus with implications on the function of possible proton channelling to a trinuclear site where the dioxygen is reduced. We solved first complex crystal structures between laccase and a phenolic substrate (2,6-dimethoxyphenol). The work revealed the binding and oxidation of phenolics in laccase-catalysed reaction. New insights from Xe binding studies were gained into how dioxygen, which is a necessity for the catalytic reaction, could be transported to the active site. 

The doctoral dissertation of M.Sc. Juha Kallio entitled Structural Studies of Ascomycete Laccases – Insights into the Reaction Pathways” will be examined at the Faculty of Science and Forestry. The opponent in the public examination is Professor Oliver Einsle, University of Freiburg and the custos is Professor Juha Rouvinen of the University of Eastern Finland.

Photo available for download at http://www.uef.fi/vaitoskuvat

Contact: Juha Kallio, tel. +358 50 410 3909, juha.kallio@uef.fi

Publishing year: 2011

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