A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

Abstract

Unspecific peroxygenases (UPO, EC 1.11.2.1) secreted by fungi open an efficient way to selectively oxyfunctionalize diverse organic substrates, including less‐activated hydrocarbons, by transferring peroxide‐borne oxygen. We investigated a cell‐free approach to incorporate epoxy and hydroxyl functionalities directly into the bulky molecule testosterone by a novel unspecific peroxygenase (UPO) that is produced by the ascomycetous fungus Chaetomium globosum in a complex medium rich in carbon and nitrogen. Purification by fast protein liquid chromatography revealed two enzyme fractions with the same molecular mass (36 kDa) and with specific activity of 4.4 to 12 U mg−1. Although the well‐known UPOs of Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) failed to convert testosterone in a comparative study, the UPO of C. globosum (CglUPO) accepted testosterone as substrate and converted it with total turnover number (TTN) of up to 7000 into two oxygenated products: the 4,5‐epoxide of testosterone in β‐configuration and 16α‐hydroxytestosterone. The reaction performed on a 100 mg scale resulted in the formation of about 90 % of the epoxide and 10 % of the hydroxylation product, both of which could be isolated with purities above 96 %. Thus, CglUPO is a promising biocatalyst for the oxyfunctionalization of bulky steroids and it will be a useful tool for the synthesis of pharmaceutically relevant steroidal molecules.

Fatty Acid Chain Shortening by a Fungal Peroxygenase

“…Since then, similar UPO proteins have been purified from other basidiomycetes and ascomycetes such as Coprinellus radians , Marasmius rotula and Chaetomium globosum ; and almost 3000 related sequences (from sequenced genomes and environmental samples) are currently available in databases . The peroxygenase from M. rotula ( Mro UPO) shows several special features compared to other UPOs, for example, the inability to oxidize halides, less pronounced capacity to oxygenate aromatics, and the unique ability for terminal hydroxylation of n ‐alkanes …”

Selective synthesis of the resveratrol analogue 4,4′-dihydroxy-trans-stilbene and stilbenoids modification by fungal peroxygenases

“…However, the main peculiarities of CglUPO's active site are: i) the absence of a conserved phenylalanine (homologous to AaeUPO Phe199, although Phe154 occupies a neighbor position) and in particular, ii) the presence of a unique and potentially reactive tyrosine (Tyr162) in the middle of the active site. Whether this tyrosine is responsible for the preferred epoxidation of isolated double bonds within complex moleculesin contrast to other UPOs, CglUPO also specifically epoxidized testosterone 18 -will have to be clarified in future mutagenesis studies. Although a yeast expression system for AaeUPO has been developed after enzyme directed evolution, 33 new procedures and hosts for expressing wild-type genes of this and other UPOs are required for investigating active-site residues and engineering UPO biocatalysts for selective oxygenation reactions of biotechnological interest.…”

“…The yield for DHS is much higher than that of chemical synthesis (only 25%) (Scheme S1 †). 18 O-labeling studies were performed in trans-stilbene reactions using H 2…”

“…18 All chromatographic steps were accomplished with an ÄKTA purifier FPLC system (GE Healthcare). The purified proteins were electrophoretically homogenous, as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions, although some minor contaminant proteins were present in CglUPO (ESI † Fig.…”

“…The highest amount of RSV (63% of products) and oxyRSV (78%) were again attained with AaeUPO. True peroxygenase activity was demonstrated by incorporation of 18 O from H 2 …”