The post-reactive structures of Leishmania major 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 Leishmania, 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 in vitro, including UDP-Glc and UDP-Gal. This enzyme, present in protozoan parasites (including Leishmania species and Trypanosoma cruzi) 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 Leishmania major USP (LmUSP) in post-reactive states. Several positions of the byproduct pyrophosphate (PPi) 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 LmUSP 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 Leishmania 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 (PPi). Here, we determined high-resolution X-ray structures of Leishmania major USP (LmUSP) 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 PPi 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.