Side chain removal from corticosteroids by unspecific peroxygenase.

Area of Science:

• Biochemistry
• Enzymology
• Fungal Biotechnology

Background:

• Unspecific peroxygenases (UPOs) are heme-containing enzymes capable of catalyzing various oxidative reactions.
• Steroids with hydroxyacetyl and hydroxyl groups at C17 are common pharmaceutical compounds and metabolic intermediates.

Purpose of the Study:

• To investigate the oxidative capabilities of UPOs from Marasmius rotula and Marasmius wettsteinii on C17-modified steroids.
• To elucidate the stepwise mechanism of steroid side-chain oxidation and fission by these enzymes.

Main Methods:

• Enzymatic oxidation assays using cortisone, Reichstein's substance S, and prednisone as substrates.
• Analysis of reaction products using chromatography and mass spectrometry.
• Protein modeling based on crystal structure data of Marasmius rotula UPO (MroUPO).
• ICP-MS analysis to confirm the presence of magnesium in purified MroUPO.

Main Results:

• UPOs from M. rotula and M. wettsteinii oxidized steroids via sequential oxygenation at C21 and C20.
• The process yielded a geminal alcohol intermediate, followed by an α-ketocarboxylic acid.
• The α-ketocarboxylic acid decomposed to adrenosterone, formic acid, and carbonic acid, without forming two-carbon fission products.
• Protein models indicated that the bulky steroid substrate fits within the MroUPO active site, allowing heme iron proximity to the steroidal side chain.
• Magnesium was detected in purified MroUPO, potentially stabilizing the porphyrin ring.

Conclusions:

• Fungal UPOs effectively metabolize steroid side chains through a defined oxidative pathway.
• The enzyme's active site architecture facilitates the oxidation of bulky steroid molecules.
• The findings contribute to understanding UPO enzymatic mechanisms and potential applications in steroid biotransformation.