High level protein-purification allows the unambiguous polypeptide determination of latent isoform PPO4 of mushroom tyrosinase ^☆

Graphical abstract

Mushroom tyrosinase diversity – six precursor isoforms, all capable of being activated and generating protein purification interfering compounds – is taking one step further to reconnaissance. This innovative method for protein purification provides access to latent tyrosinase from natural sources and enabled experiments revealing insights in a hitherto uncharacterized isoform.

Highlights

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  An innovative method for protein purification from natural sources.

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  Indications that full length PPO4 has in vivo a membrane orientated origin.

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  Mass spectrometric measurements provide exact cleavage sites for maturation.

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  Revealing PTMs in the protein backbone and strain specific sequence heterogeneity.

Abstract

Tyrosinases catalyze two initial reaction steps in the formation of melanin. Purification of tyrosinases had always been a process accompanied with various problems caused by enzymatic browning processes. Here, an approach is presented for the purification of the latent enzyme from mushrooms which averts and removes interfering compounds (e.g. polyphenols) in advance to the extraction process. The described method is supposed being well suitable as a general protein purification protocol from natural sources like fungi and plants.

The purified enzyme was investigated in detail by means of mass spectrometry: its intact protein mass was determined as 64,247.3 Da and it was identified as number four of in total six isoforms (PPO1–6) by means of sequence analysis. Some PTMs, strain specific sequence disparities and several cleavage sites including the one causing enzyme-activation (Ser ³⁸³) were determined, thus, providing insights on the maturation process of this latent tyrosinase zymogen. Based on these sequence data it can be concluded that the polypeptide backbone of the latent form of the tyrosinase PPO4 ranges from Ser ² to Thr ⁵⁶⁵, missing when compared to the gene-derived sequence a small part (46 amino acids) of the C-terminal tail. The high content on hydrophobic amino acids within this missing tail gives rise to speculations whether this part might have a function as a membrane anchor.