Structurally homologous sialidases exhibit a commonality in reactivity: Glycoside hydrolase-catalyzed hydrolysis of Kdn-thioglycosides

Aspergillus fumigatus is one of the main causative agents of invasive aspergillosis, an often-lethal fungal disease that affects immunocompromised individuals. A. fumigatus produces a sialidase that cleaves the nine-carbon carbohydrate Kdn from glycoconjugates. This enzyme plays a critical role in A. fumigatus pathogenicity, and is thus a target for the development of new therapeutics. In order to understand the reactivity of this Kdnase, and to develop a sensitive and selective assay for its catalytic activity we determined whether, like its close structural homolog the excreted sialidase produced by Micromonospora viridifaciens, this enzyme can efficiently hydrolyze thioglycoside substrates. We synthesized a panel of seven aryl 2-thio-d-glycero-α-d-galacto-non-2-ulopyranosonides and measured the activity of the A. fumigatus Kdnase towards these substrates. Four of these substrates were hydrolyzed by the A. fumigatus enzyme, although M. viridifaciens sialidase-catalyzed the hydrolysis of these Kdn thioglycosides with higher catalytic efficiencies (k_cat/K_m). We also tested an enzyme that was evolved from MvNA to improve its activity against Kdn glycosides (Glycobiology 2020, 30, 325). All three enzymes catalyzed the hydrolysis of the four most reactive Kdn thioglycosides and their second-order rate constants (k_cat/K_m) display a concave downwards Brønsted plot. The kinetic data, for each enzyme, is consistent with a change in rate-limiting step from CS bond cleavage for thioglycosides in which the pK_a of the corresponding aryl thiol is >3.6, to a non-chemical step, which is likely a conformational change, that occurs prior to CS bond cleavage for the 2,3,4,5,6-pentafluorothiophenyl glycoside.