Trypanosoma brucei, 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 T. brucei’s nucleoside diphosphate kinase B (TbNDPK), a key enzyme in nucleotide homeostasis. Circular dichroism and fluorescence spectroscopy revealed that TbNDPK is highly stable under thermal and chemical stress and undergoes nucleotide-induced conformational changes. This study also presents high-resolution crystal structures of the apo 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 TbNDPK 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.
Makori et al. (Mon,) studied this question.