Understanding the molecular basis of functional and structural stability of peroxygenases

Background.

Heme containing enzymes can function as highly versatile biocatalysts for a myriad of reactions. From the four heme peroxidase superfamilies known so far, the peroxidase-peroxygenase superfamily is the least studied despite the great variety of catalyzed reactions, ranging from peroxidase and halogenation activities to oxyfunctionalizations. In contrast to cytochromes P450 these oxidoreductases use hydrogen peroxide as cosubstrate and do not need an additional regeneration enzyme, which is an advantage for industrial applications and various other processes.
A heme-thiolate peroxygenase from Aspergillus niger has been expressed, purified and initially characterized in our lab. This is the perfect basis for further in-depth kinetic, biochemical and biophysical investigations of selected variants. Phylogenetic analyses revealed further promising targets to be investigated comparatively and in parallel.
There are still important shortcomings that have to be addressed to convert unspecific peroxygenases (UPOs) from a promising tool for biocatalysis into potent applied biocatalysts. Poor levels of heterologous expression have to be overcome, as well as the presence of undesired peroxidase activities, stability issues and oxidative self-inactivation. This project aims at building a profound basis in understanding heme-thiolate peroxygenases from A. niger and from thermophile Ascomycota (e.g. Myceliophthora thermophila) to the fullest. Digging deep into understanding substrate specificities, inactivation mechanisms, kinetic parameters of various oxyfunctionalization activities, redox potentials, structural constraints, conformational stability, and the identification of reactive intermediates during turnover will pay off to rationalize future engineering approaches.
Within this project the underlying mechanisms of specific, biotechnologically promising reactions will be investigated. By understanding the interplay between substrates, redox-cofactor and catalytically relevant amino acid residues the foundation for specific and targeted engineering will be granted. This will be achieved by structural and functional means using state-of-the-art biochemical, biophysical and structure-solving methods on the heterologously expressed and purified protein.

Research Objectives and Methods.

• understand established catalytic activities
• determine all kinetic parameters of turnover and inhibition
• assess substrate specificity
• minimize/shut down peroxidase activity
• connect findings with alternative and innovative H2O2 supply