Characterisation of the Structure and Activity of an Artificial Peroxygenase expressed in Yeast and Bacteria

The oxyfunctionalisation of inert bonds in synthetic chemistry is a very desirable yet challenging process. Methods for this activation, especially for asymmetric products, often require harsh conditions, use valuable metals or need complex catalyst synthesis. Biocatalysis may provide a more favourable approach because reactions can be carried out under mild conditions and can be highly selective. A group of heme-thiolate proteins called unspecific peroxygenases (UPOs), can carry out a diverse range of different oxyfunctionalisations using only hydrogen peroxide as a cosubstrate. The reactions carried out by UPOs are regio-, chemo- and enantioselective and they have a large substrate scope. In this work two UPOs were chosen for further investigation: an artificial UPO (artUPO) based on the UPO found in Marasmius rotula (MroUPO), and the UPO derived from Coprinus cinerea (CciUPO). The genes for artUPO and CciUPO were cloned for Pichia pastoris expression but only artUPO resulted in successful purified recombinant protein. The gene for artUPO was also successfully expressed in Escherichia coli. Both forms of artUPO were evaluated by UV/visible activity assays which displayed a small reduction in activity compared to MroUPO. The structure of both artUPO forms were determined by x-ray crystallography which developed our understanding of the mutations present in artUPO and enhanced understanding of general UPO structural biology. Substrate screens identified artUPO as effective biocatalyst for the asymmetric oxygenation of alkyl benzenes, styrenes and thioethers. With the latter, artUPO gives the complementary series of (S)-enantiomers to those obtained with the UPO from Agrocybe aegerita (AaeUPO), broadening the scope for application of the enzymes.