Broadening the Catalytic Scope of the Peroxygenase Activity of a Bacterial Tyrosine Hydroxylase

Fungal unspecific peroxygenases (UPOs, EC 1.11.2.1) are known for their unique biocatalytic properties and have been extensively studied since their discovery more than two decades ago. UPOs have found a wide range of applications in various fields, such as bioremediation, drug metabolism, and synthetic organic chemistry. Nonetheless, the production of UPOs seems limited to fungal expression systems which makes enzyme engineering challenging. It would be attractive to have a peroxygenase available that is easily expressed in a bacterial host. Here, we report on the expression and engineering of a bacterial peroxygenase, tyrosine hydroxylase (TyrH, EC 1.11.2.6). With only two rounds of computer-aided engineering, we have identified distal and active-site mutations that improve enzymatic activities and substrate scope, respectively. The combination of these mutations in a single variant led to an effective peroxygenase, capable of indigo production and enantioselective sulfoxidation and which can be overexpressed in Escherichia coli.