Abstract

Prephenate dehydrogenase (PD) catalyzes the oxidative decarboxylation of prephenate in one of the last steps of tyrosine biosynthesis. Currently all knowledge of the catalytic mechanism of this enzyme is based on a few detailed studies of the bifunctional enzyme, chorismate mutase-prephenate dehydrogenase (CM-PD), from E. coli . The putative gene for Aquifex aeolicus PD was cloned from genomic DNA and the protein overexpressed in E. coli with a removable N-terminal hexahistidine tag. The enzyme displayed Michaelis-Menten kinetics at 55C̕. A. aeolicus PD was extremely thermally stable, exhibiting maximal activity at V95C̕ with a half-life of V3 hours. Variable temperature far-UV circular dichroism and FT-infrared spectroscopies confirmed that the protein retained its secondary structure, which is mainly Ì-helical, even at high temperatures, although the protein appeared to "relax" into its active conformation with increasing temperature. The thermophilic PD was inhibited in an allosteric fashion by the end product of the pathway, tyrosine. However, size exclusion chromatography suggested that it did not undergo an oligomerization in the presence of this ligand under conditions that promoted such changes in the bifunctional E. coli enzyme. Sequence alignments with other PDs were used to identify conserved functional motifs and residues which may be important for the catalytic mechanism of the enzyme. Mutagenesis studies have been initiated at these residues, as have crystallization trials on the wild-type enzyme. Work presented in this thesis ultimately will allow comparison of the structure and enzymatic properties of the thermophilic PD to that of its mesophilic counterparts, and will contribute to the understanding of the mechanism and biochemical features of this enzyme family