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          Institute: MPI für medizinische Forschung     Collection: Abteilung Biomolekulare Mechanismen     Display Documents



ID: 307453.0, MPI für medizinische Forschung / Abteilung Biomolekulare Mechanismen
Biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus
Translation of Title:Biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus
Authors:Bonvin, Julie; Aponte, Raphael A.; Marcantonio, Maria; Singh, Sasha; Christendat, Dinseh; Turnbull, Joanne L.
Language:English
Date of Publication (YYYY-MM-DD):2006-06-01
Title of Journal:Protein Science
Journal Abbrev.:Protein Sci
Volume:15
Start Page:1417
End Page:1432
Review Status:Peer-review
Audience:Experts Only
Intended Educational Use:No
Abstract / Description: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 ({Delta}19PD) also expressed recombinantly in E. coli. Both forms are dimeric and show maximum activity at 95˚C or higher. {Delta}19PD is more sensitive to temperature effects yielding a half−life of 55 min at 95˚C versus 2 h for PD, and values of kcat and Km 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.
Free Keywords:hyperthermophile; prephenate dehydrogenase; kinetic parameters; fluorescence and CD studies; thermal unfolding
Last Change of the Resource (YYYY-MM-DD):--
External Publication Status:published
Document Type:Article
Communicated by:Wulf Kaiser
Affiliations:MPI für medizinische Forschung/Abteilung Biomolekulare Mechanismen
Identifiers:LOCALID:6650
URI:http://www.proteinscience.org/cgi/reprint/15/6/141...
URI:http://www.proteinscience.org/cgi/content/full/15/...
URI:http://www.proteinscience.org/cgi/content/abstract...
DOI:10.1110/ps.051942206
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