Structural and Functional Analyses of <i>Trypanosoma brucei</i> Nucleoside Diphosphate Kinase

<i>Trypanosoma brucei</i>, the causative agent of Human African Trypanosomiasis (HAT), relies exclusively on purine salvage for nucleotide biosynthesis, making its nucleotide-processing enzymes attractive drug targets. Here, we present a comprehensive structural and functional characterization of <i>T. brucei</i>'s nucleoside diphosphate kinase B (<i>Tb</i>NDPK), a key enzyme in nucleotide homeostasis. Circular dichroism and fluorescence spectroscopy revealed that <i>Tb</i>NDPK is highly stable under thermal and chemical stress and undergoes nucleotide-induced conformational changes. This study also presents high-resolution crystal structures of the <i>apo</i> enzyme and complexes with UDP, CDP, and GDP, showing a conserved hexameric fold, with induced-fit binding via a flexible loop involving Phe59 and key active-site residues. Enzymatic assays revealed substrate preferences for UDP and GDP, while deoxyribonucleotide diphosphates were processed with significantly reduced efficiency. Molecular dynamics simulations revealed ligand-dependent flexibility and subunit-specific nucleotide dynamics, indicating potential asymmetry and cooperative communication within the hexamer. Collectively, these findings position <i>Tb</i>NDPK as a thermostable, catalytically efficient, and structurally distinct enzyme optimized for ribonucleotide metabolism and support its potential as a selective target for future antitrypanosomal drug discovery.