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A Sialidase Mutant Displaying trans-Sialidase Activity

Abstract : Trypanosoma cruzi, the agent of Chagas disease, expresses a modified sialidase, the trans-sialidase, which transfers sialic acid from host glycoconjugates to beta-galactose present in parasite mucins. Another American trypanosome, Trypanosoma rangeli, expresses a homologous protein that has sialidase activity but is devoid of transglycosidase activity. Based on the recently determined structures of T.rangeli sialidase (TrSA) and T.cruzi trans-sialidase (TcTS), we have now constructed mutants of TrSA with the aim of studying the relevant residues in transfer activity. Five mutations, Met96-Val, Ala98-Pro, Ser120-Tyr, Gly249-Tyr and Gln284-Pro, were enough to obtain a sialidase mutant (TrSA(5mut)) with trans-sialidase activity; and a sixth mutation increased the activity to about 10% that of wild-type TcTS. The crystal structure of TrSA(5mut) revealed the formation of a trans-sialidase-like binding site for the acceptor galactose, primarily defined by the phenol group of Tyr120 and the indole ring of Trp313, which adopts a new conformation, similar to that in TcTS, induced by the Gln284-Pro mutation. The transition state analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which inhibits sialidases but is a poor inhibitor of trans-sialidase, was used to probe the active site conformation of mutant enzymes. The results show that the presence of a sugar acceptor binding-site, the fine-tuning of protein-substrate interactions and the flexibility of crucial active site residues are all important to achieve transglycosidase activity from the TrSA sialidase scaffold.
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Contributor : Marie de TARRAGON Connect in order to contact the contributor
Submitted on : Wednesday, February 17, 2021 - 5:12:42 PM
Last modification on : Thursday, April 7, 2022 - 10:10:29 AM

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Gastón Paris, Laura Ratier, María Fernanda Amaya, Tong Nguyen, Pedro M. Alzari, et al.. A Sialidase Mutant Displaying trans-Sialidase Activity. Journal of Molecular Biology, 2005, 345 (4), pp.923-934. ⟨10.1016/j.jmb.2004.09.031⟩. ⟨pasteur-03144591⟩



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