Metabolons are non-covalent complexes of enzymes that play crucial roles in primary and secondary metabolism. Such supramolecular complexes carry out a series of reactions in a pathway while protecting intermediates from diffusion into the bulk phase and promoting product channeling. This mechanism increases reaction efficiency and fidelity and protects labile intermediates. Although there has been tremendous progress in understanding the structures and functions of soluble metabolons, the study of membrane-associated metabolons is complicated by the extra dimensions added by membrane and lipidic substrates. A membrane- associated metabolon of significance is the eukaryotic dolichol pathway in which the dolichol diphosphate-linked glycan for asparagine (N)-linked of glycosylation is assembled. Bacterial glycoproteins in the epsilon proteobacteria including Campylobacter, are also generated through stepwise, membrane-associated pathways and culminate in the biosynthesis of important virulence-associated glycoproteins. Study of the bacterial pathway and extensive genomic information from Campylobacter, providing data on the enzymes and substrates in vivo across >50 discrete genera, adds a important dimension to our studies as operon order is highly conserved but, glycan output varies and there is considerable sequence divergence. Development of systematic approaches for investigating the pgl pathways will provide a valuable template for future studies on important multistep biological processes occurring at the membrane. Despite the importance of such pathways, our understanding of how the enzymes function, and whether as a metabolon or in a distributed fashion with diffusion of substrates/products between enzymes is limited. It is also unknown how the GT-B fold, common to most pgl glycosyltransferases (GTs) leads to function and substrate selectivity, as the similarity of active-site residues does not lead to a clear structure/function view, or account for the impact of the interaction of enzymes or substrates with the membrane. The research has three aims. In Aim 1, PglA, J, H1 and H2 from the C. concisus glycan assembly metabolon will be investigated via X-ray structure determination of complexes with UDP-sugar and PrenPP-sugar substrates, ligand binding and kinetic analysis, MD simulations and membrane association determination in model membrane (styrene maleic acid lipoparticles). In Aim 2, we will predict and validate protein-protein and protein-membrane interactions, determine the effect of protein-protein interactions on pathway flux and test for product channeling. Aim 3 will generate sequence similarity networks and genome neighborhood diagrams to identify orthologs in divergent species to identify structural elements governing membrane and membrane-bound substrate interactions. We will also investigate why a GT-A fold (GT) is recruited to perform the ultimate biosynthetic step. If successful, the research will provide a newly proposed ...