Mutation of Schizophyllum commune Metacaspases for the Prevention of Autocatalysis

Abstract

Metacaspases are cysteine proteases found in fungi, plants, and protozoa that contain a cysteine‐histidine dyad in their active site and cleave after lysine and arginine residues. Unlike their caspase relatives, they are not found in animals, making them an ideal target for antifungals that would incur less harm to the host. Three types of metacaspases (I, II, and III) have been characterized based on their domain structure. This work focusses specifically on the five Type I metacaspases discovered in the fungus Schizophyllum commune, which have been expressed with and without their prodomains and termed MC1‐5 and MC1‐5Δpro, respectively. Many metacaspases rely on calcium for optimal proteolytic activity, and it has also been observed that some of these metacaspases undergo autoproteolysis, or self‐cleavage, during the expression and purification process. This has posed a great challenge to our lab as the autoproteolysis of these enzymes inhibits our ability to purify them in their full length form, which is necessary in order to determine a crystal structure of the protein for structure‐function analysis. In order to purify full length enzymes, we have used site‐directed mutagenesis to create mutants that lack the cleavage sites of the wild‐type enzyme. A mutant of the MC1Δpro enzyme has been created that lacks identified cleavage sites at R389 and R447. This mutant shows significantly less of the cleavage observed in the wild‐type enzyme over a 24‐hour incubation period. Future work will focus on the identification and mutation of conserved or additional cleavage sites in the other four identified metacaspases.