The post-reactive structures of <i>Leishmania major</i> UDP-sugar pyrophosphorylase provide insights into the product release mechanism

Biosynthesis of the nucleotide sugars UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) is intimately connected and essential for the viability of trypanosomatid parasites. In the genus <i>Leishmania</i>, it is controlled by the UDP-glucose pyrophosphorylase (UGP) and UDP-sugar pyrophosphorylase (USP). In contrast to UGP, USP has a broad substrate specificity and may generate several UDP-sugars <i>in vitro,</i> including UDP-Glc and UDP-Gal. This enzyme, present in protozoan parasites (including <i>Leishmania</i> species and <i>Trypanosoma cruzi</i>) and in plants, most likely plays a role in salvaging monosaccharides. In order to gain a detailed mechanistic understanding of USPs, we determined high-resolution X-ray structures of <i>Leishmania major</i> USP (<i>Lm</i>USP) in post-reactive states. Several positions of the byproduct pyrophosphate (PP<i>i</i>) were identified and revealed a product release channel in the forward reaction, as well as the geometries of post-reactive Michaelis product complexes. The conformational changes of functional loops (hinge loop-1, hinge loop-2, and the nucleotide-binding loop) showed dynamic effects accompanying the product release process. Structural information about the post-reactive states of <i>Lm</i>USP also includes the metastable binding position of a magnesium (Mg²⁺) ion in the active site. The proposed product release mechanism was substantiated by molecular dynamics simulations and can serve as a model for other UDP-sugar pyrophosphorylases.IMPORTANCETo survive in the hostile environment of the sandfly gut, the parasite <i>Leishmania</i> relies on a range of phosphoglycans made of mannose-phosphate and galactose. In these glucose-limiting conditions, mannogen potentially serves as a reservoir for the synthesis of these crucial glycoconjugates, whereas galactose likely arises from recycling. The enzyme UDP-sugar pyrophosphorylase (USP) is responsible for the activation of this monosaccharide. This enzyme has a relaxed specificity and converts UTP and a range of sugar-1-phosphate to the corresponding UDP-sugar and pyrophosphate (PP<i>i</i>). Here, we determined high-resolution X-ray structures of <i>Leishmania major</i> USP (<i>Lm</i>USP) in post-reactive states. The data provide insight into the product release mechanism for UDP-sugar pyrophosphorylases. Considering the conservation of the residues involved in the coordination of PP<i>i</i> amongst USP enzymes, this mechanism is relevant for all USPs. This work completes our knowledge of the catalytic mechanism of trypanosomatid uridylyltransferases, which are genetically validated drug targets.