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  Biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus

Bonvin, J., Aponte, R. A., Marcantonio, M., Singh, S., Christendat, D., & Turnbull, J. L. (2006). Biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus. Protein Science, 15(6), 1417-1432. doi:10.1110/ps.051942206.

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Genre: Journal Article
Alternative Title : Biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus

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 Creators:
Bonvin, Julie, Author
Aponte, Raphael A.1, Author              
Marcantonio, Maria, Author
Singh, Sasha, Author
Christendat, Dinseh, Author
Turnbull, Joanne L., Author
Affiliations:
1Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              

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Free keywords: hyperthermophile; prephenate dehydrogenase; kinetic parameters; fluorescence and CD studies; thermal unfolding
 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.

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Language(s): eng - English
 Dates: 2005-10-312006-03-012009-01-012006-06-01
 Publication Status: Published in print
 Pages: 16
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 Table of Contents: -
 Rev. Type: Peer
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Title: Protein Science
Source Genre: Journal
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Publ. Info: New York, N.Y. : Cambridge University Press
Pages: - Volume / Issue: 15 (6) Sequence Number: - Start / End Page: 1417 - 1432 Identifier: ISSN: 0961-8368
CoNE: https://pure.mpg.de/cone/journals/resource/954925342760