Undecaprenyl Pyrophosphate Synthase (UPPS) is an enzyme that catalyzes the production of bactoprenyl diphosphate, which serves as a lipid scaffold for the biosynthesis of crucial bacterial sugars. These bacterial polysaccharides contribute to the virulence of many bacteria, but also play a role in symbiotic interactions with humans. While broad spectrum antibiotics show poor differentiation between beneficial and harmful bacteria, targeting UPPS as a narrow spectrum antibiotic allows us to select for a specific group of bacteria. UPPS is an attractive antibacterial target as it works at the start of the bacterial polysaccharide biosynthetic pathway. We are interested in probing for precise specificity between UPPS from different types of bacteria. Here, investigation into the differences between three different model organisms: the pathogenic Vibrio vulnificus, the symbiotic Bacteroides fragilis, and the typically benign Escherichia coli will allow us to distinguish structural differences to selectively target pathogenic bacteria in the future. Two fluorescent substrate analogues were used to ascertain differences in activity between each bacterial UPPS through a novel plate assay and a HPLC fluorescence based assay. These assays revealed that substrate selectivity of UPPS is bacterial species dependent. The interactions between the analogues and the active site were analyzed further in computational studies. The UPPS structure from six different organisms was used for template-based homology modeling of B. fragilis and V. vulnificus UPPS. Each was evaluated with Quantified Stability/Flexibility Relationships and molecular dynamics simulation. After generating structures for all three proteins, a docking study was performed and two residues were studied further though site directed mutagenesis. In vitro and in silico testing of mutant V. vulnificus UPPS proteins is underway to explore long range cooperativity within UPPS and understand UPPS selectivity.

Novel selective targeting of UPPS from pathogenic, symbiotic, and benign bacteria through structure, analogs, and dynamics

247th National Meeting (2014)

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