A peptide inducing self-cleavage reaction initiates the activation of tyrosinase.

The conversion of inactive pro-polyphenol oxidases (pro-PPOs) to the active enzyme results from the proteolytic cleavage of its C-terminal domain. Herein, a peptide mediated cleavage process that activates pro-MdPPO1 (Malus domestica) is reported. Mass spectrometry, mutagenesis studies and X-ray crystal structure analysis of pro-MdPPO1 (1.35 Å) and two separated C-terminal domains, one obtained upon self-cleavage (Ccleaved) of the pro-MdPPO1 (1.35 Å) and a second one produced independently (Csole) (1.05 Å), were applied to identify the structural features for the observed self-cleavage. The results reveal that the sequence Lys355-Val370 located in the linker between the active and the C-terminal domain is indispensable for the self-cleaving as a mutant lacking this peptide did not undergo self-cleavage. Partial introduction (Lys352-Ala360) of this peptide into the sequence of two PPOs, MdPPO2 and aurone synthase (CgAUS1) triggered self-cleavage in the resulting mutants. This is the first experimental proof of a self-cleavage inducing peptide in PPOs unveiling a novel mode of activation for this enzyme class that is independent of any external protease.