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 C-S 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 C–S bond cleavage for the 2,3,4,5,6-pentafluorothiophenyl glycoside.

烟曲霉是侵袭性曲霉病的主要病原体之一，这是一种影响免疫功能低下个体的常致死真菌病。A. fumigatus产生一种唾液酸酶，可从糖缀合物中裂解九碳碳水化合物 Kdn。这种酶在A. fumigatus致病性中起关键作用，因此是开发新疗法的目标。为了了解这种 Kdnase 的反应性，并对其催化活性进行灵敏和选择性的测定，我们确定了这种酶是否与其紧密结构同源物绿色小单孢菌产生的分泌唾液酸酶一样，可以有效地水解硫糖苷底物。我们合成了一组七芳基 2-硫代-d -甘油-α- d -半乳糖-non-2-ulopyranosonides 并测量烟曲霉Kdnase 对这些底物的活性。这些底物中的四种被烟曲霉酶水解，尽管M. viridifaciens唾液酸酶以更高的催化效率 ( k _cat / K _m )催化这些 Kdn 硫糖苷的水解。我们还测试了一种从Mv NA进化而来的酶，以提高其对 Kdn 糖苷的活性 (Glycobiology 2020, 30, 325)。所有三种酶都催化了四种最具反应性的 Kdn 硫糖苷的水解，它们的二级速率常数 ( k _cat / K _m ) 显示出向下凹的 Brønsted 图。对于每种酶，动力学数据与限速步骤的变化一致，从硫糖苷的 CS 键断裂（其中相应的芳基硫醇的 p K _a > 3.6）到非化学步骤，这可能是构象变化，发生在 2,3,4,5,6-五氟噻吩糖苷的 C-S 键断裂之前。