Nucleoside diphosphate kinases (NDK) are ubiquitous enzymes that catalyse the transfer of the γ phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphate (NDPs), to maintain appropriate NTP levels in cells. NDKs are associated with signal transduction, cell development, proliferation, differentiation, tumor metastasis, apoptosis and motility. The critical role of NDK in bacterial virulence renders it a potential drug target. The present manuscript reports crystal structure and functional characterization of Vibrio cholerae NDK (VNDK). The 16 kDa VNDK was crystallized in a solution containing 30% PEG 4000, 100 mM Tris-HCl pH 8.5 and 200 mM sodium acetate in orthorhombic space group P2₁2₁2₁ with unit cell parameters a = 48.37, b = 71.21, c = 89.14 Å, α = β = γ = 90° with 2 molecules in asymmetric unit. The crystal structure was solved by molecular replacement and refined to crystallographic R_factor and R_free values of 22.8% and 25.8% respectively. VNDK exists as both dimer and tetramer in solution as confirmed by size exclusion chromatography, glutaraldehyde crosslinking and small angle X-ray scattering while the crystal structure appears to be a dimer. The biophysical characterization states that VNDK has kinase and DNase activity with maximum stability at pH 8–9 and temperature up to 40 °C. VNDK shows elevated thermolability as compared to other NDK and shows preferential binding with GTP rationalized using computational studies.

核苷二磷酸激酶 (NDK) 是普遍存在的酶，可催化 γ 磷酸从三磷酸核苷 (NTP) 转移到二磷酸核苷 (NDP)，以维持细胞中适当的 NTP 水平。NDK与信号转导、细胞发育、增殖、分化、肿瘤转移、细胞凋亡和运动有关。NDK 在细菌毒力中的关键作用使其成为潜在的药物靶点。本手稿报告了霍乱弧菌NDK (VNDK) 的晶体结构和功能表征。16 kDa VNDK 在含有 30% PEG 4000、100 mM Tris-HCl pH 8.5 和 200 mM 乙酸钠的溶液中结晶，正交空间群 P2 ₁ 2 ₁ 2 ₁具有晶胞参数a  = 48.37，b  = 71.21，c  = 89.14 Å，α = β = γ = 90°，其中 2 个分子处于不对称单元中。通过分子置换求解晶体结构，并细化至晶体学R_因子和R_自由值分别为22.8%和25.8%。VNDK 在溶液中以二聚体和四聚体的形式存在，通过尺寸排阻色谱、戊二醛交联和小角 X 射线散射证实，而晶体结构似乎是二聚体。生物物理特性表明，VNDK 具有激酶和 DNase 活性，在 pH 8-9 和高达 40 °C 的温度下具有最大的稳定性。与其他 NDK 相比，VNDK 显示出更高的耐热性，并显示出与使用计算研究合理化的 GTP 的优先结合。