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