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Glycobiology, 2001, Vol. 11, No. 4 305-311
© 2001 Oxford University Press

Probing molecular function of trypanosomal sialidases: single point mutations can change substrate specificity and increase hydrolytic activity

Gaston Paris1,3, Maria Laura Cremona1,3, Maria Fernanda Amaya4, Alejandro Buschiazzo4, Susana Giambiagi4, Alberto C.C. Frasch3 and Pedro M. Alzari2,4

3Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, CC30, 1650 San Martín, Argentina, and 4Unité de Biochimie Structurale, CNRS URA 2185, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France

Sialidases are present on the surface of several trypanosomatid protozoan parasites. They are highly specific for sialic acid linked in {alpha}-(2,3) to a terminal ß-galactose and include the strictly hydrolytic enzymes and trans-sialidases (sialyl-transferases). Based on the structural comparison of the sialidase from Trypanosoma rangeli and the trans-sialidase from T. cruzi (the agent of Chagas’ disease in humans), we have explored the role of specific amino acid residues sought to be important for substrate specificity. The substitution of a conserved tryptophanyl residue in the two enzymes, Trp312/313-Ala, changed substrate specificity, rendering the point mutants capable to hydrolyze both {alpha}-(2,3)- and {alpha}-(2,6)-linked sialoconjugates. The same mutation abolished sialyl-transferase activity, indicating that transfer (but not hydrolysis) requires a precise orientation of the bound substrate. The exchange substitution of another residue that modulates oligosaccharide binding, Gln284-Pro, was found to significantly increase the hydrolytic activity of sialidase, and residue Tyr119 was confirmed to be part of a second binding site for the acceptor substrate in trans-sialidase. Together with the structural information, these results provide a consistent framework to account for the unique enzymatic properties of trypanosome trans-sialidases.

1 These authors contributed equally to this work.

2 To whom correspondence should be addressed


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