title: 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.
authors.affiliation: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, CONICET-UNSAM, CC30, 1650, San Martín, Buenos Aires, Argentina. gparis@iib.unsam.edu.ar
authors.forename: Gastón
authors.initials: G
authors.lastname: Paris
authors.forename: Laura
authors.initials: L
authors.lastname: Ratier
authors.forename: María Fernanda
authors.initials: MF
authors.lastname: Amaya
authors.forename: Tong
authors.initials: T
authors.lastname: Nguyen
authors.forename: Pedro M
authors.initials: PM
authors.lastname: Alzari
authors.forename: Alberto Carlos C
authors.initials: AC
authors.lastname: Frasch
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006023:Glycoproteins; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014345:Trypanosoma / Q000201:enzymology* / Q000235:genetics
chemical_list: D004791:Enzyme Inhibitors; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; C036137:2-deoxy-2,3-dehydro-N-acetylneuraminic acid; C069715:trans-sialidase; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid