The Crystal Structure of Aquifex aeolicus Prephenate Dehydrogenase Reveals the Mode of Tyrosine Inhibition

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).Open in a separate windowFIGURE 1.A, metabolic routes from chorismate leading to the synthesis of l-tyrosine and l-phenylalanine. In the arogenate, 4-hydroxyphenylpyruvate, or phenylpyruvate route, prephenate and arogenate are branch point intermediates in both l-tyrosine and l-phenylalanine biosynthesis. Prephenate dehydrogenase catalyzes the oxidative decarboxylation of prephenate with NAD⁺ to produce hydroxyphenylpyruvate, NADH, and CO₂ (40). B, a comparison of the chemical structure of the three ligands, HPP, HPpropionate, and tyrosine, used in the crystallization of A. aeolicus prephenate dehydrogenase. These ligands all have an -OH at the C4 position and a propionyl side chain at the C1 position of the ring.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, TyrA enzymes are highly regulated by various control mechanisms, including feedback inhibition, and genetic regulation by the Tyr operon (13-16). In some cases, l-tyrosine competes directly with substrate, be it prephenate or l-arogenate for the active site of arogenate or cyclohexadienyl dehydrogenases (14, 17-19). The product HPP can also serve as an efficient competitive inhibitor with respect to prephenate (20). Additionally, at the protein level PDs are only shown to be regulated at distinct allosteric sites or domains to modulate their activity. For example, the results of kinetic studies on the bifunctional Escherichia coli chorismate mutase-prephenate dehydrogenase (CM-PD) have indicated that this enzyme likely possesses a distinct allosteric site for binding tyrosine (21). In contrast, the Bacillus subtilis PD is the only enzyme reported to be competitively inhibited by HPP and l-tyrosine but is also noncompetitively inhibited by l-phenylalanine and l-tryptophan (12, 22). Additional regulatory control is thought to originate through a C-terminal aspartate kinase-CM-TyrA domain of the B. subtilis PD (23).Biochemical analyses of PD from E. coli CM-PD have provided a framework for understanding the molecular mechanism of the TyrA enzymes. The E. coli PD-catalyzed reaction proceeds though a rapid equilibrium, random kinetic mechanism with catalysis as the rate-limiting step (24). Additionally, studies of the pH dependence of the kinetic parameters V and V/K indicate that a deprotonated group facilitates hydride transfer from prephenate to NAD⁺ by polarizing the 4-hydroxyl group of prephenate, whereas a protonated residue is required for binding prephenate to the enzyme·NAD⁺ complex (25). The conserved residues His-197 and Arg-294 have been identified through extensive mutagenesis studies to fulfill these two roles (26, 27). Further analyses of the activities of wild-type protein and site-directed variants in the presence of a series of inhibitory substrate analogues support the idea that Arg-294 binds prephenate through the ring carboxylate (26).The structures of AD from Synechocystis sp. and PD from Aquifex aeolicus (both in complex with NAD⁺) have been reported by Legrand et al. (28) and by our group (29), respectively. Analyses of these structures have provided structural information on the conserved histidine and arginine residues. The structure A. aeolicus PD has also led to the identification of other active site residues that may play a role in enzyme catalysis, most notably Ser-126, which we propose facilitates catalysis by orienting the catalytic histidine and the nicotinamide moiety of NAD⁺ into their catalytically efficient conformations. Ambiguities can arise from examination of the binary complexes, because prephenate has only been modeled in the active site. For example, analysis of the AD structure by Legrand et al. (28) places Arg-217 (equivalent to Arg-294 in E. coli and Arg-250 in A. aeolicus) too far from the active site to play a role in prephenate binding. Thus, the full complement of interactions between prephenate and TyrA proteins are still largely unknown, as are the interactions of the enzymes with l-tyrosine.To further investigate the importance of residues involved in ligand binding, specificity, and catalysis, we have carried out co-crystallization studies of A. aeolicus PD with NAD⁺ and prephenate, with NAD⁺ and 4-hydroxyphenylpropionate (HPpropionate), a product analogue, and with NAD⁺ and l-tyrosine. Accordingly, this study provides the first direct evidence that l-tyrosine binds to the active site of a prephenate dehydrogenase. We have investigated the role of Ser-126, His-147, His-217, and Arg-250 through the kinetic analysis of site-directed mutants and structural analysis of the co-crystal complexes. To understand the role of active site residues in substrate selectivity, comparative structural analysis of AD and PD was also conducted. The current study provides a basis for understanding the mechanism of substrate selectivity between the different classes of TyrA enzymes and details how A. aeolicus PD can accept prephenate as substrate and l-tyrosine as a competitive inhibitor.