Kinetic and Structural Characterization of Sialidases (Kdnases) from Ascomycete Fungal Pathogens.

Sialidases catalyze the release of sialic acid from the terminus of glycan chains. We previously characterized the sialidase from the opportunistic fungal pathogen, and showed that it is a Kdnase. That is, this enzyme prefers 3-deoxy-d-glycero-d-galacto-non-2-ulosonates (Kdn glycosides) as the substrate compared to -acetylneuraminides (Neu5Ac). Here, we report characterization and crystal structures of putative sialidases from two other ascomycete fungal pathogens, (S) and (S). Unlike Kdnase (S), hydrolysis with the Neu5Ac substrates was negligible for S and S; thus, S and S are selective Kdnases. The second-order rate constant for hydrolysis of aryl Kdn glycosides by S is similar to that by S but 30-fold higher by S. The structures of these glycoside hydrolase family 33 (GH33) enzymes in complex with a range of ligands for both S and S show subtle changes in ring conformation that mimic the Michaelis complex, transition state, and covalent intermediate formed during catalysis. In addition, they can aid identification of important residues for distinguishing between Kdn and Neu5Ac substrates. When , and were grown in chemically defined media, Kdn was detected in mycelial extracts, but Neu5Ac was only observed in or extracts. The C8 monosaccharide 3-deoxy-d--oct-2-ulosonic acid (Kdo) was also identified in and samples. A fluorescent Kdn probe was synthesized and revealed the localization of S in vesicles at the cell surface.