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 (D19PD) also expressed recombinantly in E. coli. Both forms are dimeric and show maximum activity at 95°C or higher. D19PD 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 mutaseprephenate dehydrogenase and other mesophilic homologs.
TyrA proteins belong to a family of dehydrogenases that are dedicated to L-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, L-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our understanding of TyrA-catalyzed reactions, we have determined the crystal structures of Aquifex aeolicus prephenate dehydrogenase bound with NAD ؉ plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or L-tyrosine and have used these structures as guides to target active site residues for site-directed mutagenesis. From a combination of mutational and structural analyses, we have demonstrated that His-147 and Arg-250 are key catalytic and binding groups, respectively, and Ser-126 participates in both catalysis and substrate binding through the ligand 4-hydroxyl group. The crystal structure revealed that tyrosine, a known inhibitor, binds directly to the active site of the enzyme and not to an allosteric site. The most interesting finding though, is that mutating His-217 relieved the inhibitory effect of tyrosine on A. aeolicus prephenate dehydrogenase. The identification of a tyrosine-insensitive mutant provides a novel avenue for designing an unregulated enzyme for application in metabolic engineering.Tyrosine serves as a precursor for the synthesis of proteins and secondary metabolites such as quinones (1-3), alkaloids (4), flavonoids (5), and phenolic compounds (5, 6). In prokaryotes and plants, these compounds are important for viability and normal development (7).The TyrA protein family consists of dehydrogenase homologues that are dedicated to the biosynthesis of L-tyrosine. These enzymes participate in two independent metabolic branches that result in the conversion of prephenate to L-tyrosine, namely the arogenate route and the 4-hydroxyphenylpyruvate (HPP) 3 routes. Although both of these pathways utilize a common precursor and converge to produce a common end-product, they differ in the sequential order of enzymatic steps. Through the HPP route, prephenate is first decarboxylated by prephenate dehydrogenase (PD) to yield HPP, which is subsequently transaminated to L-tyrosine via a TyrB homologue (8). Alternatively, through the arogenate route, prephenate is first transaminated to L-arogenate by prephenate aminotransferase and then decarboxylated by arogenate dehydrogenase (AD) to yield L-tyrosine (9 -11) (see Fig. 1A).There are three classes of TyrA enzymes that catalyze the oxidative decarboxylation reactions in these two pathways. The enzymes are distinguished by the affinity for cyclohexadienyl substrates. PD and AD accept prephenate or L-arogenate, respectively, whereas the cyclohexadienyl dehydrogenases can catalyze the reaction using either substrate (12).To ensure efficient metabolite distribution of the pathway intermediates, T...
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