RESUMEN
In many prokaryotes, the first step of threonine metabolism is catalysed by the enzyme threonine dehydrogenase (TDH), which uses NAD+ to oxidize its substrate to 2-amino-3-ketobutyrate. The absence of a functional TDH gene in humans suggests that inhibitors of this enzyme may have therapeutic potential against pathogens which are reliant on this enzyme. Here, TDH from Clostridium difficile has been cloned and overexpressed, and the X-ray structure of the apoenzyme form has been determined at 2.6â Å resolution.
Asunto(s)
Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Clostridioides difficile/química , Clostridioides difficile/genética , Infección Hospitalaria , Difracción de Rayos X/métodos , Secuencia de Aminoácidos , Cristalografía por Rayos X/métodos , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Two of the world's most neglected tropical diseases, human African trypanosomiasis (HAT) and Chagas disease, are caused by protozoan parasites of the genus Trypanosoma. These organisms possess specialized metabolic pathways, frequently distinct from those in humans, which have potential to be exploited as novel drug targets. This study elucidates the structure and function of L-threonine-3-dehydrogenase (TDH) from T. brucei, the causative pathogen of HAT. TDH is a key enzyme in the metabolism of L-threonine, and an inhibitor of TDH has been shown to have trypanocidal activity in the procyclic form of T. brucei. TDH is a nonfunctional pseudogene in humans, suggesting that it may be possible to rationally design safe and specific therapies for trypanosomiasis by targeting this parasite enzyme. As an initial step, the TDH gene from T. brucei was expressed and the three-dimensional structure of the enzyme was solved by X-ray crystallography. In multiple crystallographic structures, T. brucei TDH is revealed to be a dimeric short-chain dehydrogenase that displays a considerable degree of conformational variation in its ligand-binding regions. Geometric simulations of the structure have provided insight into the dynamic behaviour of this enzyme. Furthermore, structures of TDH bound to its natural substrates and known inhibitors have been determined, giving an indication of the mechanism of catalysis of the enzyme. Collectively, these results provide vital details for future drug design to target TDH or related enzymes.