Species Differences in Alternative Substrate Utilization by the Antibacterial Target Undecaprenyl Pyrophosphate Synthase

Undecaprenyl pyrophosphate synthase (UPPS) is a critical enzyme required for the biosynthesis of polysaccharides essential for bacterial survival. In this report, we have tested the substrate selectivity of UPPS derived from the mammalian symbiont Bacteroides fragilis, the human pathogen Vibrio vulnificus, and the typically benign but opportunistic pathogen Escherichia coli. An anthranilamide-containing substrate, 2-amideanilinogeranyl diphosphate (2AA-GPP), was an effective substrate for only the B. fragilis UPPS protein, yet replacing the amide with a nitrile [2-nitrileanilinogeranyl diphosphate (2CNA-GPP)] led to a compound that was fully functional for UPPS from all three target organisms. These fluorescent substrate analogues were also found to undergo increases in fluorescence upon isoprenoid chain elongation, and this increase in fluorescence can be utilized to monitor the activity and inhibition of UPPS in 96-well plate assays. The fluorescence of 2CNA-GPP increased by a factor of 2.5-fold upon chain elongation, while that of 2AA-GPP increased only 1.2-fold. The 2CNA-GPP compound was therefore more versatile for screening the activity of UPPS from multiple species of bacteria and underwent a larger increase in fluorescence that improved its ability to detect increases in chain length. Overall, this work describes the development of new assay methods for UPPS and demonstrates the difference in substrate utilization between forms of UPPS from different species, which has major implications for UPPS inhibitor development, assay construction, and the development of polysaccharide biosynthesis probes.