Validation and characterization of two targets for drug design: The polyamine pathway's role in growth and biofilm formation in Yersinia pestis; and poly(ADP -ribose) glycohydrolase, an enzyme involved in DNA repair in mammals

In this dissertation evidence was gathered to validate two' metabolic pathways as targets for drug design.;Part I of this dissertation investigates small cationic polymers known as polyamines and their biosynthesis in bacteria as a potential target for antibacterial drug design. Polyamines represent ubiquitous organic cations found in virtually every life form in which they have been investigated. Polyamines bind nucleic acid, proteins and lipids, modulating their function. The polyamine pathway of the bacterium Yersinia pestis was investigated using gene deletion mutants of two key enzymes in the biosynthetic pathway, ornithine decarboxylase (ODC) and arginine decarboxylase (ADC). These mutants were characterized in terms of their growth and biofilm forming properties and complemented both genetically and chemically. The biofilm-investigation showed a dominant effect by ADC in Y. pestis biofilm formation. Key polyamine transporters and biosynthetic enzymes in Y. pestis and other pathogenic biofilm forming bacteria were mapped using sequence alignments. The sequence analysis identified key enzymes that could be targeted by genetic or chemical means to characterize growth and biofilm effects upon polyamine depletion. Y. pestis ADC was enzymatically and structurally characterized. ADC has unique enzymatic requirements for pyridoxal 5' phosphate (PLP) and Mg2+ for activity and potentially unique protein structural features. ADC has been overexpressed, and crystallized. Preliminary X-ray crystallographic data was collected. In addition, a novel link shows a direct correlation between the levels of the polyamine putrescine and biofilm formation.;Part II of this dissertation focuses on poly(ADP-ribose) (PAR) found primarily in multicellular eukaryotic organisms. This polymer is negatively charged and its presence strongly correlates with genomic stability. There are 18 known poly(ADP-ribose) polymerases (PARP) that polymerize nicotinamide adenosine dinucleotide (NAD) into poly(ADP-ribose), but currently only one enzyme, poly(ADP-ribose) glycohydrolase (PARG) is known to break down the polymer. PARG activity is required for cell survival and is a unique target for anticancer drug design. Key PARG amino acids were rationally mutated and analyzed for binding and catalytic effects. The catalytic site and binding pocket amino acids of PARG were identified and a functional sequence fingerprint for genomic searches was identified.