High level protein-purification allows the unambiguous polypeptide determination of latent isoform PPO4 of mushroom tyrosinase.
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 (Ser³⁸³) 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 Ser² to Thr⁵⁶⁵, 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.
Department of Biophysical Chemistry, University of Vienna, Althanstraße 14, 1090 Vienna, Austria.
Stephan G
SG
Mauracher
Department of Biophysical Chemistry, University of Vienna, Althanstraße 14, 1090 Vienna, Austria.
Christian
C
Molitor
Department of Analytical Chemistry, University of Vienna, Währinger Straße 38, 1090 Vienna, Austria.
Claudia
C
Michael
Department of Biophysical Chemistry, University of Vienna, Althanstraße 14, 1090 Vienna, Austria.
Martin
M
Kragl
Department of Analytical Chemistry, University of Vienna, Währinger Straße 38, 1090 Vienna, Austria.
Andreas
A
Rizzi
Department of Biophysical Chemistry, University of Vienna, Althanstraße 14, 1090 Vienna, Austria. Electronic address: annette.rompel@univie.ac.at.
Annette
A
Rompel
D000363:Agaricales / Q000201:enzymology*; D055162:Biocatalysis; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010455:Peptides / Q000032:analysis* / Q000378:metabolism
D007527:Isoenzymes; D010455:Peptides; D014442:Monophenol Monooxygenase
Agaricus bisporus; Latent tyrosinase; Metalloenzyme; PPO4; Polyphenols; Post translational modification; Protease inhibitor; Type-3 copper center; Tyrosinase maturation; Zymogen