Aspergillus fumigatus infections are rising at a disconcerting rate in tandem with antifungal resistance rates. Efforts to develop novel antifungals have been hindered by the limited knowledge of fundamental biological and structural mechanisms of A. fumigatus propagation. Biosynthesis of NTPs, the building blocks of DNA and RNA, is catalysed by NDK. An essential enzyme in A. fumigatus, NDK poses as an attractive target for novel antifungals. NDK exhibits broad substrate specificity across species, using both purines and pyrimidines, but the selectivity of such nucleosides in A. fumigatus NDK is unknown, impeding structure‐guided inhibitor design. Structures of NDK in unbound‐ and NDP‐bound states were solved, and NDK activity was assessed in the presence of various NTP substrates. We present the first instance of a unique substrate binding mode adopted by CDP and TDP specific to A. fumigatus NDK that illuminates the structural determinants of selectivity. Analysis of the oligomeric state reveals that A. fumigatus NDK adopts a hexameric assembly in both unbound‐ and NDP‐bound states, contrary to previous reports suggesting it is tetrameric. Kinetic analysis revealed that ATP exhibited the greatest turnover rate (321 ± 33.0 s⁻¹), specificity constant (626 ± 110.0 mm⁻¹·s⁻¹) and binding free energy change (−37.0 ± 3.5 kcal·mol⁻¹). Comparatively, cytidine nucleosides displayed the slowest turnover rate (53.1 ± 3.7 s⁻¹) and lowest specificity constant (40.2 ± 4.4 mm⁻¹·s⁻¹). We conclude that NDK exhibits nucleoside selectivity whereby adenine nucleosides are used preferentially compared to cytidine nucleosides, and these insights can be exploited to guide drug design.

中文翻译：

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烟曲霉核苷二磷酸激酶的核苷选择性

烟曲霉感染与抗真菌药耐药率呈令人不安的上升趋势。由于对烟曲霉繁殖的基本生物学和结构机理的了解有限，阻碍了开发新型抗真菌药的努力。NDK催化NTP的生物合成，DNA和DNA的组成部分。NDK是烟曲霉中的必不可少的酶，是新型抗真菌药的诱人靶标。NDK在嘌呤和嘧啶中均表现出跨物种的广泛底物特异性，但这类核苷酸在烟曲霉中的选择性NDK未知，阻碍了结构导向抑制剂的设计。解决了未结合和未结合NDP的NDK结构，并在存在各种NTP底物的情况下评估了NDK的活性。我们呈现了独特的底物结合模式的第一个实例，该模式由烟曲霉NDK特有的CDP和TDP采用，阐明了选择性的结构决定因素。对低聚状态的分析表明，烟曲霉NDK在未结合和与NDP结合状态均采用六聚体组装，这与以前的报道表明它是四聚体相反。动力学分析表明，ATP具有最大的周转率（321±33.0 s ^-1），特异性常数（626±110.0 m m ^-1 ·s ^-1））和结合自由能变化（-37.0±3.5 kcal·mol ^-1）。相比之下，胞苷核苷显示出最慢的周转率（53.1±3.7 s ^-1）和最低的特异性常数（40.2±4.4 m m ^-1 ·s ^-1）。我们得出结论，NDK具有核苷选择性，因此相比胞苷核苷，优先使用腺嘌呤核苷，这些见解可用于指导药物设计。