Researcher(s)
- Sofia Forgione, Biochemistry, University of Delaware
Faculty Mentor(s)
- Catherine Grimes, Chemistry & Biochemistry, University of Delaware
Abstract
The human microbiome is responsible for a multitude of essential bodily functions, including immune system regulation, defense, digestion, and more. This includes the function of the innate immune system, which is governed by peptidoglycan (PG), the macromolecule that forms the bacterial cell wall. PG contains a glycan backbone comprised of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), connected via a β-(1,4) glycosidic linkage and cross-linked by a pentapeptide chain. Understanding each aspect of the PG biosynthesis pathway is crucial for treating various autoimmune diseases involving disruption of the human microbiome, such as Crohn’s Disease. This includes PG recycling, which will be explored by purifying the key enzymes involved and examining their activity and substrate specificity. This process is explored in detail by the Grimes Lab, identifying AmgK, MurU, MurC, and MurD as essential enzymes in a salvage pathway for the synthesis of PG intermediates in commensals and pathogenic bacteria. Here, the purification and characterization of these proteins from Pseudomonas aeruginosa, a major human pathogen, was optimized. Each of these enzymes was obtained and subsequently used in a large-scale synthesis and purification of UDP intermediates such as UDP-NAM and UDP-MDP. Purification was achieved using high-performance liquid chromatography (HPLC), and the results were analyzed by LC-MS and NMR. A successful large-scale chemoenzymatic synthesis of both naturally occurring UDP sugars and those modified with an azide handle was achieved, with yields greater than 33.33%. These UDP sugars will then be used to probe the promiscuity of O-linked NAG transferase, the sole glycosyltransferase in O-GlcNAcylation. Preliminary data suggest that the bacterial UDP-sugars can stabilise the OGT, providing new connections between mammalian enzymes and bacterial PG fragments. Ultimately, this work used traditional biochemistry to deliver novel substrates to probe human physiology.