Bioorg Chem. 2026 Jun 30;180:110193. doi: 10.1016/j.bioorg.2026.110193. Online ahead of print.

ABSTRACT

Biocatalytic CH bond activation offers a sustainable approach for the selective transformation of inert hydrocarbons. Unspecific peroxygenase (UPOs) are cofactor-free alternatives to P450 monooxygenase, capable of performing diverse oxygenation reactions on a broad range of substrates. In this study, we characterized a hypothetical unspecific peroxygenase from the fungus Daldinia sp. (DspUPO-I), which efficiently catalyzes the hydroxylation of medium- to long-chain fatty acids, primary alcohols, and short-chain alkanes. The enzyme was heterologously expressed in Escherichia coli, and structurally characterized by X-ray crystallography. To enhance DspUPO-I activity and selectivity, the residues around binding site were analyzed using docking and molecular dynamics simulations, followed by structure-guided mutagenesis of neighboring sites to identify the key residues involved and to elucidate their role in aliphatic hydroxylation. Notably, single mutation at Ile53 and Leu208 generated variants I53F and L208H with a twofold enhanced activity. Furthermore, modification of Ala163, yielding variant A163C and A163I with 50-90% selectivity toward subterminal hydroxylation of carboxylic acids and primary alcohols. In addition, combination of mutations at Phe53, Ala163, and Leu208 increased enzyme’s regioselectivity, although the activities were lower than that of wild-type (wt) DspUPO-I. Analysis of mutants’ structure revealed their narrower active site channel than wt, which restricted substrate binding access, increasing enzyme activity and regioselectivity. These findings provide structural insight of DspUPO-I and highlight its potential as a robust biocatalyst for selective oxyfunctionalization of aliphatic CH bonds under mild conditions.

PMID:42385391 | DOI:10.1016/j.bioorg.2026.110193