Structural conservation and functional divergence in a tyrosinase from Paenibacillus polymyxa with a flexible C-terminal domain

Tyrosinases are type-3 copper enzymes that are widely distributed across various forms of life. These enzymes feature a conserved binuclear copper active center that is responsible for oxidizing both monophenols and diphenols. In this study, a tyrosinase-like protein from Paenibacillus polymyxa was heterologously produced and characterized. Sequence analysis and AlphaFold3 modeling identified a conserved catalytic domain at the N-terminus with a typical type-3 copper center coordinated by six histidine residues in a four-helix bundle and a C-terminal domain. The purified enzyme was found to contain 1.9 ± 0.1 copper ions per polypeptide chain, and presented the characteristic absorption bands at 345 nm, suggesting the presence of a μ-η²:η² peroxo-bridged copper center. Circular dichroism spectroscopy confirmed the mixed α/β fold of the protein consistent with its structural model and revealed its high thermal stability, with a melting temperature of 73 ± 1 °C. Cyclic voltammetry experiments identified a redox process with a formal potential of +279 ± 15 mV versus NHE, which is consistent with the reduction of the peroxo bridge in the active site. Despite its conserved structural features, the isolated enzyme displayed low catalytic efficiency (0.042 s^-1·mM^-1) in the oxidation of L-Dopa. These findings indicate that structural diversification within the type-3 copper protein family can substantially alter enzymatic activity, illustrating the evolutionary and functional diversity of tyrosinases and related enzymes.