Genomic Enzymology to Decipher Bacterial Membrane-Associated Glycan Assembly

Summary Glycoconjugates are critical for bacterial survival and virulence, as they play key roles in cell-wall integrity, immune evasion, and host-pathogen interactions. The biosynthesis of many bacterial glycoconjugates is achieved via an “en bloc” transfer strategy. Biosynthesis is initiated by membrane-embedded polyprenol phosphate (PrenP) phosphoglycosyl transferases (PGTs), that catalyze the first membrane-committed step, which involves the transfer of a phosphosugar from a nucleoside diphosphate (NDP)-activated donor to an amphiphilic PrenP acceptor. This step affords a membrane-bound PrenPP-sugar product, which is then elongated by the action of a cadre of glycosyltransferases (GTs) that define each new glycosidic linkage to “code” the new glycan. The assembled PrenPP-glycan is then transferred across the membrane by a flippase and ultimately the glycan is transferred to protein, lipid or glycan acceptors. In prokaryotes, NDP-sugar substrates display greater variation compared to eukaryotes, which significantly broadens the diversity of glycoconjugate products in microorganisms. The proposed work will to identify and assign signatures for substrate specificity in PGT and GT enzymes such that the glycan composition can ultimately be inferred at the protein sequence level. Moreover, the monotopic PGT (monoPGT) superfamily is exclusive to prokaryotic, allowing for the development of selective inhibitors towards at crucial survival and virulence targets without affecting eukaryotic systems. Our recent research has shed light on the monoPGT structure, dynamics, and membrane topology. We have developed a sequence similarity network with isofunctional groupings that provide sequence motifs for functional assignment across the monoPGT superfamily. Analysis of the products and structures of GTs from Campylobacter has allowed identification of specificity determinants for their cognate UDP-sugar substrate. Changes in sugar specificity are found to be reflected in the evolutionary divergence of members later in the pathway. Future research aims to understand enzyme specificity through genomic enzymology, focusing on pathways from pathogens. We will apply bioinformatics, biochemical analysis, and structural biology approaches to connect primary sequence information to UDP-sugar substrate specificity for the monoPGT, GT-B and GT-A superfamilies. The GT structures allow the pursuit of computational studies of the dynamics of GT-substrate complexes to assess membrane topology and enzyme conformation as a specificity determinant and possible effector of processivity. Structures of the bifunctional (Bi) monoPGT family members combined with informatics will shed light on protein:protein interactions between pathway enzymes and provide insight into metabolon formation in glycan biosynthesis. We propose leveraging structure prediction to identify membrane-associated GT-Bs to uncover new metabolic pathways for glycan biosynthesis. Ultimately, integration of computational and experimental studies will further elucidate the interactions between enzymes, membranes, and substrates, uncovering new pathways and regulatory mechanisms critical to bacterial survival and pathogenesis. Project Number: 1R35GM161405-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Karen Allen | Institution: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS), BOSTON, MA | Award Amount: $437,036 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MBBC-A (57)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11259968