Production of N-terminal-less metacaspase from Candida albicans containing site-directed mutations: Importance of the calcium-interaction site for activation and control of enzyme activity.

• Grant number: 25/04605-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: January 01, 2026
• End date: December 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Mauricio Ferreira Marcondes Machado
• Grantee: Aline Felipe Araujo
• Host Institution: Pró-Reitoria Acadêmica. Universidade de Mogi das Cruzes (UMC). Campus da Sede Mogi das Cruzes. Mogi das Cruzes , SP, Brazil

Abstract

This research project aims to produce Candida albicans metacaspase (CaMCA-¿N86) containing site-directed mutations in the aspartate residues responsible for the interaction of calcium with this enzyme. The CaMCA-¿N86 metacaspase undergoes self-processing to become catalytically active, and requires calcium ion as an enzymatic cofactor both for its self-processing and for the performance of its enzymatic activity. For this reason, we intend to produce this enzyme containing site-directed mutations in the four aspartate residues responsible for the interaction of CaMCA-¿N86 with calcium, namely Asp252, Asp268, Asp269 and Asp299, and thus try to elucidate the importance of calcium in the self-processing and regulation of the activity of this protease. Previous studies performed by our group have shown that the mutation of a single residue demonstrated that only 3 of the 4 aspartate residues demonstrated in the 3D structure of CaMCA-¿N86 affect the interaction of Ca2+ with this enzyme, but none are crucial for the binding of this cofactor. For this reason, we intend to produce this enzyme containing site-directed mutations in the four aspartate residues exchanging them for alanine, but we will produce a triple mutant (D268/269/299A), in addition to producing a mutant of the four aspartate residues (CaMCA-¿N86Ca-Null), in order to try to elucidate the importance of calcium in the self-processing and regulation of the activity of this protease. Such a study may contribute to the rational design of molecules that can inhibit and/or activate CaMCA-¿N86, being of great importance for the development of possible new drugs. (AU)