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Identification of Key Active‐Site Positions Controlling the Chemoselectivity of Aspergillus Brasiliensis Unspecific Peroxygenase
Author(s) -
Schmitz Fabian,
Hoffrogge Maike,
Koschorreck Katja,
Fukuta Yasuhisa,
Raffaele Alessandra,
Tieves Florian,
Hilberath Thomas,
Hollmann Frank,
Urlacher Vlada B.
Publication year - 2025
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202500181
Heme‐containing unspecific peroxygenases (UPOs) have attracted significant attention as biocatalysts for oxidation reactions due to their ability to function without expensive nicotinamide cofactors. In the recent study, the UPO from aspergillus brasiliensis ( Abr UPO) is found to catalyze the aromatic hydroxylation of substituted benzenes, a feature that distinguishes Abr UPO from other reported wild‐type UPOs. To elucidate the underlying factors in the active site and substrate access channel of Abr UPO—which contains fewer phenylalanine residues compared to other UPOs that primarily catalyze benzylic hydroxylation—twenty two Abr UPO variants with single, double, triple, or quadruple amino acid substitutions were constructed to mimic the active sites or substrate access channels of other UPOs. A number of mutated variants exhibited altered activity and selectivity, and several positions were identified that influence enzyme chemoselectivity. Among them, substitution of alanine at position 186 with bulkier residues such as phenylalanine or leucine lead to a shift in chemoselectivity toward alkyl chain hydroxylation of substituted benzenes. Molecular docking studies indicated that the A186F mutation restricts the flexibility and reorientation of ethylbenzene in the active site of Abr UPO, thereby preventing oxidation at the aromatic ring while promoting benzylic hydroxylation.

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