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Biochemical and biophysical characterization of a prephenate dehyodrogenase from the hyperthermophilic bacterium Aquifex aeolicus


Biochemical and biophysical characterization of a prephenate dehyodrogenase from the hyperthermophilic bacterium Aquifex aeolicus

Bonvin, Julie (2008) Biochemical and biophysical characterization of a prephenate dehyodrogenase from the hyperthermophilic bacterium Aquifex aeolicus. PhD thesis, Concordia University.

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Prephenate dehydrogenase is a key enzyme from the TyrA protein family responsible for catalyzing the NAD+ -dependent oxidative decarboxylation of prephenate to hydroxylphenylpyruvate, one of the terminal steps in the biosynthesis of tyrosine (Tyr). To gain structural and biophysical information on this protein, PD from the thermophilic bacterium Aquifex aeolicus was expressed as a His-tagged protein in Escherichia coli and was purified by nickel affinity chromatography. The enzyme is susceptible to proteolysis at the N-terminal region of the protein and the exact site of cleavage was determined by mass spectrometry. Crystallography trials on several N-terminally truncated variants performed by our collaborators at the University of Toronto indicated that only the PD variant missing the first 19 amino acids (E19PD) yielded quality diffraction crystals. The biochemical and biophysical properties of the full-length PD were compared to E19PD also expressed recombinantly in E. coli . The enzyme functions as a cyclohexadienyl dehydrogenase, accepting prephenate (effectively) and L-arogenate (poorly) as substrates. Both forms of the enzyme are thermally stable and show maximal activity only at high temperatures, although E19PD is less stable but more active than the full-length protein. Low concentrations of the denaturant guanidinium hydrochloride (Gdn-HCl) activate the activity of E19PD, but at higher concentrations activity is lost concomitant with a multi-state pathway of denaturation which 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 (Trp) residues per monomer, one is buried in a hydrophobic pocket and does not become solvent exposed until the protein unfolds, while the less buried Trp is at the active site. These findings are in accordance with the crystal structure of E19PD. Site-directed mutagenesis and steady-state kinetic analyses of variant proteins were used to probe the roles of conserved residues. In accord with the crystal structure of the enzyme bound with NAD+ plus product and product analogues, His147 acted as a catalytic hydrogen bond acceptor while Ser216 was responsible for coordinating NAD+ and His147 to facilitate hydride transfer. Arg250 and His217 were responsible for binding prephenate in the active site. Additionally and most importantly, His217 in A. aeolicus PD and the homologous residue in E. coli CM-PD (His257) was shown to be critical for inhibition of activity by Tyr. Two assays were developed to assess Tyr binding to wild-type and variant enzymes. Our results are placed in context of crystal structures of PD bound with Tyr and indicate how TyrA proteins can accept hydroxyphenylpuruvate and Tyr in the active site of the enzyme.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (PhD)
Authors:Bonvin, Julie
Pagination:xviii, 207 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:Ph. D.
Thesis Supervisor(s):Turnbull, J
ID Code:975227
Deposited By: Concordia University Library
Deposited On:22 Jan 2013 15:44
Last Modified:18 Jan 2018 17:39
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