Related Experiment Videos
A sialidase mutant displaying trans-sialidase activity
Gastón Paris1, Laura Ratier, María Fernanda Amaya
1Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, CONICET-UNSAM, CC30, 1650, San Martín, Buenos Aires, Argentina. gparis@iib.unsam.edu.ar
Journal of Molecular Biology
|December 14, 2004
Summary
Researchers engineered Trypanosoma rangeli sialidase (TrSA) to gain trans-sialidase activity, crucial for Chagas disease. Key mutations created a binding site for sugar acceptors, enabling transfer activity from a sialidase scaffold.
Area of Science:
- Biochemistry
- Structural Biology
- Parasitology
Background:
- Trypanosoma cruzi causes Chagas disease, utilizing trans-sialidase (TcTS) to transfer sialic acid.
- Trypanosoma rangeli possesses a homologous sialidase (TrSA) lacking transglycosidase activity.
Purpose of the Study:
- To engineer TrSA to exhibit trans-sialidase activity by identifying key residues.
- To understand the structural basis for transglycosidase activity in sialidases.
Main Methods:
- Site-directed mutagenesis of TrSA based on structural data of TrSA and TcTS.
- Crystallography to determine the structure of engineered mutants.
- Enzyme activity assays using transition state analogues.
Main Results:
- Five mutations (Met96-Val, Ala98-Pro, Ser120-Tyr, Gly249-Tyr, Gln284-Pro) conferred trans-sialidase activity to TrSA.
- A sixth mutation further enhanced activity to ~10% of wild-type TcTS.
- Crystal structure revealed a TcTS-like acceptor binding site in the mutant.
Conclusions:
- Engineering TrSA into a trans-sialidase is achievable through targeted mutations.
- Formation of a specific acceptor binding site and conformational flexibility are critical for transglycosidase function.
- This work provides insights into the evolution and mechanism of sialidases and trans-sialidases.