UDP-glucose (UDP-Glc) is synthesized by UGP2-encoded UDP-Glc pyrophosphorylase (UGP) and is required for glycoconjugate biosynthesis and galactose metabolism because it is a uridyl donor for galactose-1-P (Gal1P) uridyltransferase. Chinese hamster lung fibroblasts harboring a hypomrphic UGP(G116D) variant display reduced UDP-Glc levels and cannot grow if galactose is the sole carbon source. Here, these cells were cultivated with glucose in either the absence or presence of galactose in order to investigate glycoconjugate biosynthesis and galactose metabolism. The UGP-deficient cells display < 5% control levels of UDP-Glc/UDP-Gal and > 100-fold reduction of [6-³H]galactose incorporation into UDP-[6-³H]galactose, as well as multiple deficits in glycoconjugate biosynthesis. Cultivation of these cells in the presence of galactose leads to partial restoration of UDP-Glc levels, galactose metabolism and glycoconjugate biosynthesis. The V_max for recombinant human UGP(G116D) with Glc1P is 2000-fold less than that of the wild-type protein, and UGP(G116D) displayed a mildly elevated K_m for Glc1P, but no activity of the mutant enzyme towards Gal1P was detectable. To conclude, although the mechanism behind UDP-Glc/Gal production in the UGP-deficient cells remains to be determined, the capacity of this cell line to change its glycosylation status as a function of extracellular galactose makes it a useful, reversible model with which to study different aspects of galactose metabolism and glycoconjugate biosynthesis.

Leishmaniasis is a neglected disease that is caused by different species of the protozoan parasite Leishmania, and it currently affects 12 million people worldwide. The antileishmanial therapeutic arsenal remains very limited in number and efficacy, and there is no vaccine for this parasitic disease. One pathway that has been genetically validated as an antileishmanial drug target is the biosynthesis of uridine diphosphate-glucose (UDP-Glc), and its direct derivative UDP-galactose (UDP-Gal). De novo biosynthesis of these two nucleotide sugars is controlled by the specific UDP-glucose pyrophosphorylase (UGP). Leishmania parasites additionally express a UDP-sugar pyrophosphorylase (USP) responsible for monosaccharides salvage that is able to generate both UDP-Gal and UDP-Glc. The inactivation of the two parasite pyrophosphorylases UGP and USP, results in parasite death. The present study reports on the identification of structurally diverse scaffolds for the development of USP inhibitors by fragment library screening. Based on this screening, we selected a small set of commercially available compounds, and identified molecules that inhibit both Leishmania major USP and UGP, with a half-maximal inhibitory concentration in the 100 µM range. The inhibitors were predicted to bind at allosteric regulation sites, which were validated by mutagenesis studies. This study sets the stage for the development of potent USP inhibitors.

In all kingdoms of life, the enzyme uridine diphosphate-glucose pyrophosphorylase (UGP) occupies a central role in metabolism, as its reaction product uridine diphosphate-glucose (UDP-Glc) is involved in various crucial cellular processes. Pathogens, including fungi, parasites, and bacteria, depend on UGP for the synthesis of virulence factors; in particular, various bacterial species utilize UDP-Glc and its derivatives for the synthesis of lipopolysaccharides, capsular polysaccharides, and biofilm exopolysaccharides. UGPs have, therefore, gained attention as anti-bacterial drug target candidates, prompting us to study their structure-function relationships to provide a basis for the rational development of specific inhibitors. UGP function is tied to its oligomeric state, and the majority of bacterial homologs have been described as tetramers encoded by the galU gene. Uniquely, enterobacterial species harbor a second gene, galF , encoding a protein with high homology to UGP, whose function is somewhat controversial. Here, we show that the galF gene of the opportunistic pathogen Klebsiella pneumoniae encodes a dimeric protein that has lost UGP activity, likely due to a combination of active site mutations and an inability to tetramerize, whereas the functional K. pneumoniae UGP, encoded by galU , is an active tetramer. Our AlphaFold-assisted structure-function relationship studies underline that tetramerization is essential for bacterial UGP function and is facilitated by a common mechanism utilizing conserved key residues. Targeting the respective molecular interfaces, which are absent in human UGP, could provide a means of selectively inhibiting the bacterial virulence factor UGP and potentially rendering pathogenic species avirulent. IMPORTANCE: The enzyme uridine diphosphate-glucose pyrophosphorylase (UGP) is important for the virulence of bacterial pathogens and, therefore, a potential drug target. In this study, we identify the gene encoding the functional UGP in Klebsiella pneumoniae , a bacterium notoriously causing severe antibiotic-resistant infections in humans, and reveal structural and functional features that may aid in the development of new antibiotics.

Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi , two pathogens causing considerable economic loss in the swine, sheep, and cattle industries.

Multidrug-resistant bacteria such as the opportunistic pathogen Pseudomonas aeruginosa , which causes life-threatening infections especially in immunocompromised individuals and cystic fibrosis patients, pose an increasing threat to public health. In the search for new treatment options, P. aeruginosa uridine diphosphate-glucose pyrophosphorylase (PaUGP) has been proposed as a novel drug target because it is required for the biosynthesis of important virulence factors and linked to pathogenicity in animal models. Here, we show that UGP-deficient P. aeruginosa exhibits severely reduced virulence against human lung tissue and cells, emphasizing the enzyme’s suitability as a drug target. To establish a basis for the development of selective PaUGP inhibitors, we solved the product-bound crystal structure of tetrameric PaUGP and conducted a comprehensive structure-function analysis, identifying key residues at two different molecular interfaces that are essential for tetramer integrity and catalytic activity and demonstrating that tetramerization is pivotal for PaUGP function. Importantly, we show that part of the PaUGP oligomerization interface is uniquely conserved across bacterial UGPs but does not exist in the human enzyme, therefore representing an allosteric site that may be targeted to selectively inhibit bacterial UGPs. IMPORTANCE: Infections with the opportunistic bacterial pathogen Pseudomonas aeruginosa are becoming increasingly difficult to treat due to multidrug resistance. Here, we show that the enzyme uridine diphosphate-glucose pyrophosphorylase (UGP) is involved in P. aeruginosa virulence toward human lung tissue and cells, making it a potential target for the development of new antibacterial drugs. Our exploration of P. aeruginosa (Pa)UGP structure-function relationships reveals that the activity of PaUGP depends on the formation of a tetrameric enzyme complex. We found that a molecular interface involved in tetramer formation is conserved in all bacterial UGPs but not in the human enzyme, and therefore hypothesize that it provides an ideal point of attack to selectively inhibit bacterial UGPs and exploit them as drug targets.

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 ( Lm USP) in post-reactive states. Several positions of the byproduct pyrophosphate (PP 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 Lm USP also includes the metastable binding position of a magnesium (Mg ²⁺ ) 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. IMPORTANCE: To 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 (PP i ). Here, we determined high-resolution X-ray structures of Leishmania major USP ( Lm 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 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.