Toolkit development for bacterial isoprenoid utilizing enzymes

? DESCRIPTION (provided by applicant): Bactoprenyl diphosphate (BPP) is a critical component in the biosynthesis of complex bacterial polysaccharides including teichoic acids and capsules, which play central roles in the virulence of many pathogenic bacteria. Undecaprenyl Pyrophosphate Synthase (UPPS) is responsible for the production of BPP and is common to all bacteria, yet the proteins responsible for polysaccharide production vary a great deal from one organism to another. To mark UPPS or polysaccharide biosynthesis proteins for inhibition rapid assays need to be available to screen the function and inhibition of these enzymes. However, appropriate assays are not available for any of these proteins, and with respect to polysaccharide biosynthesis proteins very few are even characterized. Polysaccharide assembly enzymes are generally not well characterized primarily due to the lack of isoprenoid-linked substrates available to study their activity. Part of the problem is that substrate is diffiult to obtain and its utilization is laborious to track in vitro. Rapid assays are available to monitorthe inhibition of UPPS, yet these methods are blind to important aspects of the reaction that could provide important mechanisms for the inhibition of the functional consequence of UPPS activity, the production of a fully functional C55 BPP. This proposal aims to develop new tools for the identification of polysaccharide assembly proteins as well as rapid throughput methods to screen their inhibition. In addition, this proposal focuses on a new method to screen UPPS activity that allows for the detection of both rates of catalysis as well as the consequence to the product formed in the presence of various inhibitors of the protein.