Summary Complex glycoconjugates play a pivotal role in bacterial survival, colonization and virulence and thus, to the interactions between symbiotic and pathogenic bacteria and their human hosts. An important mechanism for the assembly of complex structures involves initiation of glycan assembly on the cytoplasmic face of cell membranes catalyzed by polyprenol phosphate (PrenP) phosphoglycosyl transferases (PGTs). PGTs catalyze transfer of a C1’-phosphosugar moiety from a soluble nucleoside diphosphate-activated donor to a PrenP acceptor, yielding a membrane-bound polyprenol diphosphosugar. The proposed studies focus on a PGT superfamily with a monotopic membrane topology for which, until our recent studies, there has been only limited structural and mechanistic information. These enzymes differ in structure, mechanism and topology from the well-known polytopic PGTs. Biochemical studies together with the recently-determined structure of Campylobacter concisus PglC from our laboratories, show that the monotopic PGTs include a reentrant membrane helix (RMH) that penetrates only one leaflet of the bilayer then re-emerges. Aim 1 will Identify sugar-specificity determinants for all three families within the monotopic PGT superfamily by determining X-ray crystal structures of liganded complexes with nucleoside diphosphate sugar substrates. This will enable assignment of specificity of newly-identified monotopic PGTs and provide information on the function of PGTs in the glycoconjugate biosynthetic pathways of various pathogens. In Aim 2 the model that binding of the UDP-sugar substrate triggers the movement of the soluble residue loop (aa 61-80) to complete substrate-binding determinants and close the active site for catalysis will be tested using cross-linking and fluorescence-based approaches. The sequence of the RMH integral to membrane interaction will be used to develop HMMs to identify similar RMH via bioinformatics within the monotopic PGT superfamily and then applied to unrelated proteins predicted to have RMHs, those potentially misannotated as bitopic and other integral membrane proteins. Aim 3 develops nucleoside derivatives that will serve as fluorescent probes, activity-based protein profiling probes and inhibitors of the monotopc PGT superfamily. Overall the in-depth study of the substrate specificities and functions of the monotopic superfamily and design of biological probes will establish the fundamental knowledge and tools needed for validating and intervening in the action of potential therapeutic targets.