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, Aspergillus fumigatus, and showed that it is a Kdnase. That is, this enzyme prefers 3-deoxy-Dglycero-D-galacto-non-2-ulosonates (Kdn glycosides) as the substrate compared to N-acetylneuraminides (NeuSAc). Here, we report characterization and crystal structures of putative sialidases from two other ascomycete fungal pathogens, Aspergillus terreus (AtS) and Trichophyton rubrum (TrS). Unlike A. fumigatus Kdnase (AfS), hydrolysis with the NeuSAc substrates was negligible for TrS and AtS; thus, TrS and AtS are selective Kdnases. The secondorder rate constant for hydrolysis of aryl Kdn glycosides by AtS is similar to that by AtS but 30-fold higher by TrS. The structures of these glycoside hydrolase family 33 (GH33) enzymes in complex with a range of ligands for both AtS and TrS 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 NeuSAc substrates. When A. fumigatus, A. terreus, and T. rubrum were grown in chemically defined media, Kdn was detected in mycelial extracts, but NeuSAc was only observed in A. terreus or T. rubrum extracts. The C8 monosaccharide 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) was also identified in A. fumigatus and T. rubrum samples. A fluorescent Kdn probe was synthesized and revealed the localization of AtS in vesides at the cell surface.