The pro-enzyme C-terminal processing domain of Pholiota nameko tyrosinase is responsible for folding of the N-terminal catalytic domain.

Pholiota nameko (Pholiota microspore) tyrosinase is expressed as a latent 67-kDa pro-tyrosinase, comprising a 42-kDa N-terminal catalytic domain with a binuclear copper centre and a 25-kDa C-terminal domain and is activated by proteolytic digestion of the C-terminal domain. To investigate the role of the C-terminal processing domain of pro-tyrosinase, we constructed a recombinant tyrosinase lacking the C-terminal domain and four recombinant pro-tyrosinase mutants (F515G, H539N, L540G and Y543G) carrying substituted amino acid residues on the C-terminal domain. The recombinant tyrosinase lacking the C-terminal domain had no catalytic activity; whereas the mutant L540G was copper depleted, the other mutants had copper contents similar to that of the wild-type pro-tyrosinase. Proteolytic digestion activated the mutants H539N and Y543G following release of the C-terminal domain, and the resulting tyrosinases had higher K m values for t-butyl catechol than the wild-type pro-tyrosinase. The mutants F515G and L540G were degraded by proteolytic digestion and yielded smaller proteins with no activity. These data suggest that the C-terminal processing domain of P. nameko pro-tyrosinase is essential for correct folding of the N-terminal catalytic domain and acts as an intramolecular chaperone during assembly of the active-site conformation.

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

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.