Discovery of Bacterial Unspecific Peroxygenases

Unspecific peroxygenases (UPOs, EC 1.11.2.1) are promising biocatalysts for the oxyfunctionalization of organic molecules and the synthesis of industrially relevant compounds because of their vast repertoire of catalyzed reactions. To date, thousands of putative UPO genes have been identified in eukaryotic genomes, most of them in the Ascomycota and Basidiomycota phyla, and several UPOs have been characterized. Remarkably, no related enzymes have been reported in prokaryotic organisms. Here, we describe the discovery of a diverse family of bacterial heme-thiolate peroxygenases through structure database mining, followed by functional characterization of selected representatives. The bacterial proteins, termed bacterial UPOs (BUPOs), display clear structural homology to family I (short) fungal UPOs despite low sequence identity (<20%). Expression of one of these proteins (HydBUPO) in its native host (Hydrogenophaga sp. A37) was confirmed by proteomics. Several BUPOs were cloned and expressed in Escherichia coli. In biochemical assays, the BUPOs were able to catalyze one-electron oxidation (peroxidase activity) of ABTS and 2,6-dimethoxyphenol, as well as two-electron oxidation (peroxygenase activity) of naphthalene, indole, 3-phenyl-1-propanol, and 16-hydroxypalmitic acid, using hydrogen peroxide as a cosubstrate. These enzymes thus represent a previously unknown group of bacterial heme-thiolate peroxygenases that share key structural and functional features with eukaryotic UPOs, offering potential candidates for the development of industrially relevant biocatalysts.