title: 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 (Delta19PD) also expressed recombinantly in E. coli. Both forms are dimeric and show maximum activity at 95 degrees C or higher. Delta19PD is more sensitive to temperature effects yielding a half-life of 55 min at 95 degrees C versus 2 h for PD, and values of kcat and Km for prephenate, which are twice those determined for PD at 80 degrees 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.
authors.affiliation: Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec H4B 1R6, Canada.
authors.forename: Julie
authors.initials: J
authors.lastname: Bonvin
authors.forename: Raphael A
authors.initials: RA
authors.lastname: Aponte
authors.forename: Maria
authors.initials: M
authors.lastname: Marcantonio
authors.forename: Sasha
authors.initials: S
authors.lastname: Singh
authors.forename: Dinesh
authors.initials: D
authors.lastname: Christendat
authors.forename: Joanne L
authors.initials: JL
authors.lastname: Turnbull
mesh_terms: D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D019281:Dimerization; D004795:Enzyme Stability; D005453:Fluorescence; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011303:Prephenate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D021122:Protein Subunits; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D021122:Protein Subunits; C076398:TyrA protein, Bacteria; D014443:Tyrosine; D014364:Tryptophan; D011303:Prephenate Dehydrogenase; D019791:Guanidine