Biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus

Abstract

A monofunctional prephenate dehydrogenase (PD) from Aquifex aeolicus was expressed as a His‐tagged protein in Escherichia coli and was purified by nickel affinity chromatography allowing the first biochemical and biophysical characterization of a thermostable PD. A. aeolicus PD is susceptible to proteolysis. In this report, the properties of the full‐length PD are compared with one of these products, an N‐terminally truncated protein variant (Δ19PD) also expressed recombinantly in E. coli . Both forms are dimeric and show maximum activity at 95°C or higher. Δ19PD is more sensitive to temperature effects yielding a half‐life of 55 min at 95°C versus 2 h for PD, and values of k cat and K m for prephenate, which are twice those determined for PD at 80°C. Low concentrations of guanidine‐HCl activate enzyme activity, but at higher concentrations activity is lost concomitant with a multi‐state pathway of denaturation that proceeds through unfolding of the dimer, oligomerization, then unfolding of monomers. Measurements of steady‐state fluorescence intensity and its quenching by acrylamide in the presence of Gdn‐HCl suggest that, of the two tryptophan residues per monomer, one is buried in a hydrophobic pocket and does not become solvent exposed until the protein unfolds, while the less buried tryptophan is at the active site. Tyrosine is a feedback inhibitor of PD activity over a wide temperature range and enhances the cooperativity between subunits in the binding of prephenate. Properties of this thermostable PD are compared and contrasted with those of E. coli chorismate mutase‐prephenate dehydrogenase and other mesophilic homologs.