Identification and characterization of the functional tetrameric UDP-glucose pyrophosphorylase from <i>Klebsiella pneumoniae</i>

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 <i>galU</i> gene. Uniquely, enterobacterial species harbor a second gene, <i>galF</i>, encoding a protein with high homology to UGP, whose function is somewhat controversial. Here, we show that the <i>galF</i> gene of the opportunistic pathogen <i>Klebsiella pneumoniae</i> 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 <i>K. pneumoniae</i> UGP, encoded by <i>galU</i>, 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.IMPORTANCEThe 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 <i>Klebsiella pneumoniae</i>, 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.