C-terminal processing of tyrosinase is responsible for activation of Pholiota microspora proenzyme

• Published in: Applied microbiology and biotechnology Vol. 90; no. 1; pp. 227 - 234
• Main Authors: Kawamura-Konishi, Yasuko, Maekawa, Saya, Tsuji, Mariko, Goto, Hideyuki
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.04.2011; Springer-Verlag; Springer; Springer Nature B.V
• Subjects: Activation; Amino Acid Motifs; Amino Acid Sequence; Applied Genetics and Molecular Biotechnology; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Cloning; E coli; Enzymatic activity; Enzyme Activation; Enzyme kinetics; Enzyme Precursors - chemistry; Enzyme Precursors - genetics; Enzyme Precursors - metabolism; Enzymes; Escherichia coli; Food; Fundamental and applied biological sciences. Psychology; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Fungi; Genetic engineering; Kinetics; Laboratories; Life Sciences; Microbial Genetics and Genomics; Microbiology; Microspora; Molecular Sequence Data; Monophenol Monooxygenase - chemistry; Monophenol Monooxygenase - genetics; Monophenol Monooxygenase - metabolism; Mushrooms; Oxidation; Pholiota - chemistry; Pholiota - enzymology; Pholiota - genetics; Pholiota microspora; Pholiota nameko; Pigments; Plasmids; Polypeptides; Proenzyme; Protein Processing, Post-Translational; Proteins; Studies; Tyrosinase; Proenzyme; Activation; Tyrosinase; Pholiota microspora; Enzyme; Oxidoreductases; Monophenol monooxygenase; Pholiota nameko
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-010-3039-8

Tyrosinase is expressed as a 67-kDa protein in Pholiota microspora (synonym Pholiota nameko), whereas the same enzyme purified from fruiting bodies of P. microspora is a 42-kDa protein that is cleaved with a C-terminal 25-kDa polypeptide from the 67-kDa protein. To confirm the role of C-terminal processing in enzyme activity, we expressed a recombinant 67-kDa tyrosinase in Escherichia coli cells. To obtain a soluble protein, the recombinant tyrosinase was expressed as a thioredoxin fusion protein with an enterokinase-cleavable site. Enterokinase digestion of the fusion protein produced a recombinant 67-kDa tyrosinase that did not have any catalytic activity. However, chymotrypsin digestion of the fusion protein produced a recombinant 44-kDa tyrosinase that was catalytically active and had a 25-kDa cleaved C-terminal. Kinetic parameters of the 44-kDa tyrosinase were similar to those of the 42-kDa tyrosinase purified from the fruiting bodies. These results suggest that tyrosinase is expressed in P. microspora as a latent 67-kDa proenzyme and is converted to the mature active 42-kDa enzyme by proteolytic processing of the C-terminal.