Identification of an Activity Selector for the Nitroso-Forming Activity in Bacterial Type-III Copper Enzymes

Abstract

O-aminophenol oxidases, a specialized subclass of type-III copper proteins, play a crucial role in the biosynthesis of bioactive nitrosophenols which display antiretroviral and blood cholesterol lowering activity. Another related subclass of type-III copper proteins, tyrosinases, closely resemble o-aminophenol oxidases both structurally and enzymatically but lack their unique ability to oxidize o-aminophenols into nitrosophenols. To unpuzzle the catalytic disparities of both subclasses, highly conserved amino acid residues in vicinity of the catalytic center were identified. Notably, the Asn43 residue in the o-aminophenol oxidase from Streptomyces griseus (SgGriF) plays a pivotal role in its nitroso-forming activity. Mutating the Asn43 residue in SgGriF to isoleucine, which is present at the homologous position Ile42 in the tyrosinase from Streptomyces sp. ZL-24 (SzTYR) resulted in the loss of the nitroso-forming activity in SgGriF. Conversely, exchanging Ile42 in SzTYR to asparagine is generating nitroso-forming activity in SzTYR. The results presented herein demonstrate the feasibility of converting an o-aminophenol oxidase into a tyrosinase and vice versa through a single amino acid mutation, underscoring the potential of these findings for future applications in medicinal and material sciences.