Institute of Photonics and Advanced Sensing (IPAS), School of Biological Sciences, The University of Adelaide, Australia.
Adelaide Proteomics Centre, School of Physical Sciences, The University of Adelaide, Australia.
FEBS J. 2021 Apr;288(7):2398-2417. doi: 10.1111/febs.15607. Epub 2020 Nov 6.
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 ), specificity constant (626 ± 110.0 mm ·s ) and binding free energy change (-37.0 ± 3.5 kcal·mol ). Comparatively, cytidine nucleosides displayed the slowest turnover rate (53.1 ± 3.7 s ) and lowest specificity constant (40.2 ± 4.4 mm ·s ). 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. ENZYMES: Nucleoside-diphosphate kinase (EC 2.7.4.6). DATABASE: Structural data are available in the PDB database under the accession numbers: Unbound-NDK (6XP4), ADP-NDK (6XP7), GDP-NDK (6XPS), IDP-NDK (6XPU), UDP-NDK (6XPT), CDP-NDK (6XPW), TDP-NDK (6XPV).
烟曲霉感染与抗真菌药物耐药率一起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制的了解有限，因此开发新型抗真菌药物的努力受到了阻碍。NTP 的生物合成，即 DNA 和 RNA 的构建块，由 NDK 催化。NDK 是烟曲霉中的一种必需酶，也是新型抗真菌药物的一个有吸引力的靶点。NDK 在物种间表现出广泛的底物特异性，既能使用嘌呤又能使用嘧啶，但烟曲霉 NDK 中此类核苷的选择性尚不清楚，这阻碍了基于结构的抑制剂设计。已解决无结合物和 NDP 结合物状态下的 NDK 结构，并评估了各种 NTP 底物存在下的 NDK 活性。我们首次展示了一种独特的 CDP 和 TDP 结合模式，该模式特定于烟曲霉 NDK，阐明了选择性的结构决定因素。寡聚状态分析表明，烟曲霉 NDK 在无结合物和 NDP 结合物状态下均采用六聚体组装，与先前报道的四聚体相反。动力学分析表明，ATP 表现出最大的周转率（321±33.0 s）、特异性常数（626±110.0 mm·s）和结合自由能变化（-37.0±3.5 kcal·mol）。相比之下，胞苷核苷显示出最慢的周转率（53.1±3.7 s）和最低的特异性常数（40.2±4.4 mm·s）。我们得出结论，NDK 表现出核苷选择性，与胞苷核苷相比，腺嘌呤核苷优先使用，这些见解可用于指导药物设计。酶：核苷二磷酸激酶（EC 2.7.4.6）。数据库：结构数据可在 PDB 数据库中获得，登录号为：无结合物-NDK（6XP4）、ADP-NDK（6XP7）、GDP-NDK（6XPS）、IDP-NDK（6XPU）、UDP-NDK（6XPT）、CDP-NDK（6XPW）、TDP-NDK（6XPV）。
