Molecular modeling of T. rangeli, T. brucei gambiense, and T. evansi sialidases in complex with the DANA inhibitor.

Trypanosomal (trans-) sialidases are enzymes that catalyze the transfer of sialic acid residues between host and parasite glycoconjugates. Herein, we have used homology modeling to construct the 3D structures of sialidases from Trypanosoma brucei and Trypanosoma evansi. Hybrid quantum mechanical/molecular mechanical molecular dynamics simulations were used to determine the interaction energy between the 2-deoxy-2,3-didehydro-N-acetylneuraminic acid inhibitor and the three sialidases studied here. Our results suggest that the two constructed enzymes share the same basic fold motive of the Trypanosoma rangeli crystallographic structure. In addition, quantum mechanical/molecular mechanical molecular dynamics simulations show that the 2-deoxy-2,3-didehydro-N-acetylneuraminic acid inhibitor forms a stronger complex with Trypanosoma rangeli than with Trypanosoma brucei and Trypanosoma evansi sialidases. Finally, the interaction energy by residues shows that the arginine triad plays a decisive role to complex 2-deoxy-2,3-didehydro-N-acetylneuraminic acid with the enzyme through hydrogen bonding.

A sialidase mutant displaying trans-sialidase activity.

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.

Specificity of the binding site of the sialic acid-binding lectin from ovine placenta, deduced from interactions with synthetic analogues.

The specificity of the sialic acid-binding lectin from ovine placenta was examined in detail by haemagglutination inhibition assays applying a panel of 32 synthetic sialic acid analogues. The carboxylic acid group is a prerequisite for the interaction with the lectin, the alpha-anomer of the methyl glycoside is only a little more effective as an inhibitor than the beta-anomer and the most potent inhibitor was 9-deoxy-10-carboxylic acid Neu5Ac, followed by 4-oxo-Neu5Ac. In contrast to the majority of known sialic acid-binding lectins, the N-acetyl group of Neu5Ac is not indispensable for binding, neither is the hydroxyl group at C-9 since substitutions at this carbon atom are well tolerated. Furthermore, all sulfur-containing substituents at C-9 enhanced the affinity of the lectin. This is the first sialic acid-binding lectin found to strongly bind thio derivatives.