Vieira Plínio S, de Jesus Santos Ana P, de Oliveira Arthur H C
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, Zip code: 14041-901, Ribeirão Preto - SP, Brazil.
Protein Pept Lett. 2016;23(2):99-106. doi: 10.2174/0929866523666151109113241.
Nucleoside diphosphate kinases (NDK; EC 2.7.4.6) are enzymes required for maintaining intracellular levels of nucleosides triphosphates (NTP) through transfer the γ-phosphoryl group from a NTP to a NDP. The enzyme is associated with several biological functions including prevention of host ATP-mediated cytolysis during pathogenic infections. Here we present the biophysical characterization of NDK from Leishmania major and the effect of a mutation on the protein structure in solution. The structural stability was analyzed since this secreted protein may act in different microenvironments at various stages of the parasite life cycle. LmNDK and P95S mutant were subjected to denaturation with pH and guanidine. Structural transitions were monitored by circular dichroism and intrinsic fluorescence tryptophan emission. Our results showed that the LmNDK is more structurally stable than other described NDKs and that the catalytically active P95S mutant in the Kpn loop presented a decrease in protein stability, indicating the importance of this proline for maintenance of the LmNDK structure.
核苷二磷酸激酶（NDK；EC 2.7.4.6）是通过将三磷酸核苷（NTP）的γ-磷酸基团转移至二磷酸核苷（NDP）来维持细胞内核苷三磷酸（NTP）水平所必需的酶。该酶与多种生物学功能相关，包括在致病性感染期间防止宿主ATP介导的细胞溶解。在此，我们展示了来自硕大利什曼原虫的NDK的生物物理特性以及溶液中一种突变对蛋白质结构的影响。由于这种分泌蛋白可能在寄生虫生命周期的不同阶段作用于不同的微环境，因此对其结构稳定性进行了分析。将硕大利什曼原虫NDK（LmNDK）和P95S突变体用pH值和胍进行变性处理。通过圆二色性和色氨酸的固有荧光发射监测结构转变。我们的结果表明，LmNDK在结构上比其他已描述的NDK更稳定，并且Kpn环中具有催化活性的P95S突变体的蛋白质稳定性降低，表明该脯氨酸对于维持LmNDK结构的重要性。
