Experimental and bioinformatic investigation of the proteolytic degradation of the C-terminal domain of a fungal tyrosinase

Proteolytic processing is a key step in the production of polyphenol oxidases such as tyrosinases, converting the inactive proenzyme to an active form. In general, the fungal tyrosinase gene codes for a ~ 60 kDa protein that is, however, isolated as an active enzyme of ~ 40 kDa, lacking the C-terminal domain. Using the secreted tyrosinase 2 from Trichoderma reesei as a model protein, we performed a mutagenesis study of the residues in proximity of the experimentally determined cleavage site which are possibly involved in the proteolytic process. However, the mutant forms of tyrosinase 2 were not secreted in a full-length form retaining the C-terminal domain, but they were processed to give a ~ 45 kDa active form. Aiming at explaining this phenomenon, we analysed in silico the properties of the C-terminal domain of tyrosinase 2, of 23 previously retrieved homologous tyrosinase sequences from fungi (C. Gasparetti, G. Faccio, M. Arvas, J. Buchert, M. Saloheimo, K. Kruus, Appl. Microbiol. Biotechnol. 86 (2010) 213–226) and of nine well-characterised polyphenol oxidases. Based on the results of our study, we exclude the key role of specific amino acids at the cleavage site in the proteolytic process and report an overall higher sensitivity to proteolysis of the linker region and of the whole C-terminal domain of fungal tyrosinases.

Active tyrosinase is often produced by removal of the C-terminal domain from a pro-enzyme. Mutagenesis and sequence analyses exclude the role of amino acids at the cleavage site in the process and report a higher level of intrinsic disorder and sensitivity to proteolysis of the whole C-terminal domain of tyrosinases.Figure optionsDownload as PowerPoint slideHighlights
► Proteolytic processing is a key step in the production of tyrosinases.
► Tyrosinase 2 from T. reesei was differently mutagenised at the C-terminal cleavage site.
► Neither YG-motif nor specific residues are critical for C-terminal processing of tyrosinase.
► The C-terminal domain carries intrinsic disorder and higher sensitivity to proteolysis.