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

Abstract

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 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 the vicinity of the catalytic center were identified. Notably, the Asn43 residue in the o ‐aminophenol oxidase from Streptomyces griseus ( Sg GriF) plays a pivotal role in its nitroso‐forming activity. Mutating the Asn43 residue in Sg GriF to isoleucine, which is present at the homologous position Ile42 in the tyrosinase from Streptomyces sp. ZL‐24 ( Sz TYR), resulted in the loss of the nitroso‐forming activity in Sg GriF. Conversely, exchanging Ile42 in Sz TYR to asparagine generates nitroso‐forming activity in Sz TYR. 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.