Abstract
Regulatory proteins from pathogenic bacteria have the potential to serve as novel therapeutic targets. This study discusses the characterization of three regulatory bacterial proteins. First, the HlyU protein of Vibrio vulnificus belongs to the SmtB/ArsR family of metal ion binding repressors and is a global regulator of virulence factors. It has been shown to directly bind a DNA sequence upstream of the transcription start site in the rtxA1 operon and to compete with the repressor H-NS. In this study, a homology model was constructed using the crystal structure of the metallorepressor SmtB from Synechococcus elongatus PCC7942. A high-throughput assay was utilized to monitor DNA binding in vitro. The model structure, metal analysis, and denaturation assays all support the hypothesis that HlyU lacks the key metal ion binding residues of other members of the SmtB/ArsR family. Future studies will determine key residues in DNA recognition and binding and will determine how HlyU is regulated. Second, the Diphtheria toxin repressor (DtxR) is the best characterized member of a family of metal ion-activated repressors involved in metal ion homeostasis, virulence factor expression, and stress response in bacteria. This study presents evidence that DtxR may be involved in an oxidative stress related response system through the oxidation of the metal ion ligand C102. Future studies will determine how, structurally, oxidation of C102 abolishes repressor activity. Third, propionate kinase (PK) from the highly infectious bacterial pathogen Francisella tularensis is a key enzyme in the metabolism of serine and threonine. Previous studies determined a 2.2 Å structure of the apo-enzyme. In this study, structural studies were performed to cocrystallize PK with its substrate in order to better understand the mechanism of phosphate transfer. While crystals with substrate were not obtained, a novel crystallization condition of the apo-enzyme was determined. Future studies will reveal the structure of propionate kinase bound to its substrate.