A Peptide-Induced Self-Cleavage Reaction Initiates the Activation of Tyrosinase

Abstract

The conversion of inactive pro-polyphenol oxidases (pro-PPOs) into 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 of pro-MdPPO1 and the other one produced independently, were applied to study the observed self-cleavage. The sequence Lys 355-Val 370 located in the linker between the active and the C-terminal domain is indispensable for the self-cleavage. Partial introduction (Lys 352-Ala 360) of this peptide into the sequence of two other 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 new mode of activation for this enzyme class that is independent of any external protease.

Keywords: activating peptides; crystal structures; maturation agents; polyphenol oxidases; self-cleaving peptides.

Figure 1

Crystal structure of pro‐MdPPO1 (PDB No. 6ELS) and the C_cleaved‐domain (PDB No. 6ELT). A) The overall structure of pro‐MdPPO1. The main domain is shown in green, the C‐terminal domain in red, and the linker that connects the main and C‐terminal domains in blue. Owing to the absence of electron density, a part (Ala 349–Val 359) of the loop region within the C‐terminal domain is missing. B) The overall structure of the C_cleaved‐domain with the Ca²⁺ binding site. Ca²⁺ (purple sphere) is coordinated by three aspartate residues (shown in stick mode) and three water molecules depicted as small red spheres.

Figure 2

The different cleavage sites of wild‐type MdPPO1, mutant‐1, and mutant‐2. The wild type is cleaved within the sequence Ser 366–Ser 367–Ser 368–Lys 369–Val 370, mutant‐1 within Lys 355–Lys 356–Lys 357, and mutant‐2 within His 361–Ala 362–Ala 363.

Figure 3

Structural comparison of MdPPO1, MdPPO2, and their respective mutants. A) Superposition of the crystal structure of MdPPO1 (green cartoon) and the homology model of MdPPO2 (magenta cartoon), which was prepared by using the SWISS‐MODEL Server.29 The black rectangle highlights a region of the linker where the isoenzymes differ significantly. The inset on the right indicates the region Ala 349–Val 359 of MdPPO1, which is missing in the structure owing to a lack of electron density and was therefore modelled with the software MODELLER28 (blue cartoon). B) The effect of the deletion of the peptide Lys 355–Val 370 (cyan cartoon) from the sequence of MdPPO1 (green cartoon) on the region highlighted in (A). The resulting mutant MdPPO1(−) does not exhibit self‐cleavage. C) The effect of the insertion of the peptide Lys 352–Ala 360 (KVAKKLGVA) from MdPPO1 (green cartoon) into the sequence of MdPPO2 (magenta cartoon) on the region highlighted in (A). The insertion converts the stable MdPPO2 into a self‐cleaving enzyme, mutant MdPPO2(+). D) Primary structures of MdPPO1, MdPPO2, and the respective mutants. The Figure highlights which part of the sequence was deleted from MdPPO1 to produce MdPPO1(−) and which sequence part was added to MdPPO2 to obtain MdPPO2(+). The black triangles indicate the respective cleavage sites in MdPPO1 and MdPPO2(+). E: glutamic acid, H: histidine, N: asparagine, D: aspartic acid, T: threonine, G: glycine, F: phenylalanine, V: valine, R: arginine, L: leucine, K: lysine, P: proline, A: alanine, S: serine, W: tryptophan.