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Kinetic and structural characterization of sialidases (Kdnases) from ascomycete fungal pathogens

DOI: 10.1021/acschembio.1c00666 DOI Help

Authors: Ali Nejatie (Simon Fraser University) , Elizabeth Steves (Simon Fraser University) , Nick Gauthier (Simon Fraser University) , Jamie Baker (Simon Fraser University) , Jason Nesbitt (Simon Fraser University) , Stephen A. Mcmahon (University of St Andrews) , Verena Oehler (University of St Andrews) , Nicholas J. Thornton (University of St Andrews) , Benjamin Noyovitz (University of British Columbia) , Kobra Khazaei (Simon Fraser University) , Brock W. Byers (Simon Fraser University) , Wesley F. Zandberg (University of British Columbia) , Tracey M. Gloster (University of St Andrews) , Margo M. Moore (Simon Fraser University) , Andrew J. Bennet (Simon Fraser University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Chemical Biology , VOL 16 , PAGES 2632 - 2640

State: Published (Approved)
Published: November 2021

Abstract: 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-d-glycero-d-galacto-non-2-ulosonates (Kdn glycosides) as the substrate compared to N-acetylneuraminides (Neu5Ac). 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 Neu5Ac substrates was negligible for TrS and AtS; thus, TrS and AtS are selective Kdnases. The second-order rate constant for hydrolysis of aryl Kdn glycosides by AtS is similar to that by AfS 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 Neu5Ac substrates. When A. fumigatus, A. terreus, and T. rubrum were grown in chemically defined media, Kdn was detected in mycelial extracts, but Neu5Ac 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 AfS in vesicles at the cell surface.

Journal Keywords: Noncovalent interactions; Carbohydrates; Peptides and proteins; Conformation; Cells

Diamond Keywords: Fungi; Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Added On: 09/05/2022 11:42

Discipline Tags:

Pathogens Infectious Diseases Health & Wellbeing Biochemistry Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)