Complex glycoconjugates play a pivotal role in bacterial survival, colonization, and virulence, and contribute to the interactions between symbiotic and pathogenic bacteria and their human hosts. Assembly of these macromolecules is initiated on the cytoplasmic face of cell membranes, catalyzed by polyprenol phosphate (PrenP) phosphoglycosyl transferases (PGTs). PGTs transfer a C1’-phosphosugar from a soluble nucleoside diphosphate-sugar to a PrenP acceptor, yielding a membrane-bound polyprenol diphosphosugar. Our studies focus on the exclusively prokaryotic PGT superfamily with a monotopic membrane topology (monoPGTs). Our work has previously led to the mechanistic and structural characterization of the monoPGTs, revealing a unique reentrant membrane helix supporting the structure of the active-site residues and substrate-binding determinants. Identification of this core fold has enabled bioinformatic analysis of sequences from diverse bacteria where the gene encoding the PGT enables identification of the “signature step” in a dedicated set of genes that, together, describe the glycan of the glycoconjugate product. The proposed studies will investigate the structures and binding landscapes of the monoPGT superfamily, and the design of biological probes will establish the fundamental knowledge and tools needed for validating and intervening in the action of potential therapeutic targets. In Aim 1, sequence similarity networks will guide the choice of candidates for X-ray crystallographic analysis that will be determined with detergent-solubilized protein in the small (Sm) monoPGTs, which encodes the core fold without elaboration. Substrate and inhibitor-liganded structures and activity analysis will elucidate the determinants of substrate specificity. Genome neighborhood networks will inform on the presence of genes in the operon that catalyze the biosynthesis of unusual sugars to be tested as substrates. Aim 2 will address the pathway regulation and flux assisted by the determination of X-ray and CryoEM structures. Protein- protein interactions will be analyzed via covariance analysis and elucidation of the structure of bifunctional (Bi) monoPGTs, fusions of monoPGTs and glycosyltransferases, which will also define membrane interactions and electrostatics. The binding of nucleotides to the proposed regulatory domain of unknown function (DUF) present in the large (Lg) monoPGTs and dehydrogenases in the pathway will be tested. Aim 3 builds on nucleoside analogs from solid-phase synthesis and non-hydrolyzable uridine bisphosphonate-sugars (UBPs) as probes and inhibitors for the prokaryotic monoPGT superfamily. The structure of UBPs bound to monoPGT targets in Aim 1 will inform further design and identification of specificity determinants. Overall, these comprehensive and in-depth studies will provide a detailed structural and functional understanding of this untapped bacterial enzyme superfamily and knowledge of their glycoconjugate pathways and cel...