Identification of a plant gene encoding glutamate/aspartate-prephenate aminotransferase: the last homeless enzyme of aromatic amino acids biosynthesis

In all organisms synthesising phenylalanine and/or tyrosine via arogenate, a prephenate aminotransferase is required for the transamination of prephenate into arogenate. The identity of the gene encoding this enzyme in the organisms where this activity occurs is still unknown. Glutamate/aspartate-prephenate aminotransferase (PAT) is thus the last homeless enzyme in the aromatic amino acids pathway. We report on the purification, mass spectrometry identification and biochemical characterization of Arabidopsis thaliana prephenate aminotransferase. Our data revealed that this activity is housed by the prokaryotic-type plastidic aspartate aminotransferase (At2g22250). This represents the first identification of a gene encoding PAT.     

A kinetic and structural comparison of chorismate mutase/prephenate dehydratase from mutant strains of Escherichia coli K12 defective in the PheA gene

Three classes of mutant strains of Escherichia coli K12 defective in pheA, the gene coding for chorismate mutase/prephenate dehydratase, have been isolated: (1) those lacking prephenate dehydratase activity, (2) those lacking chorismate mutase activity, and (3) those lacking both activities. Chorismate mutase/prephenate dehydratase from the second class of mutants was less sensitive to inhibition by phenylalanine than wild-type enzyme and, along with the defective enzyme from the third class of mutants, could not be purified by affinity chromatography on Sepharosyl-phenylalanine. Pure chorismate mutase/prephenate dehydratase protein was prepared from two strains belonging to the first class. The chorismate mutase activity of these enzymes is kinetically similar to that of the wild-type enzyme except for a two- to threefold increase in both the K_a for chorismate and the K_is for inhibition by prephenate. In both cases only one change in the tryptic fingerprint was detected, resulting from a substitution of the threonine residue in the peptide Gln·Asn·Phe·Thr·Arg. This suggests that this residue is catalytically or structurally essential for the dehydratase activity.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: spatial relationship of the mutase and dehydrogenase sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase (4-hydroxyphenylpyruvate synthase) by substrate analogues has been investigated at pH 6.0 with the aim of elucidating the spatial relationship that exists between the sites at which each reaction occurs. Several chorismate and adamantane derivatives, as well as 2-hydroxyphenyl acetate and diethyl malonate, act as linear competitive inhibitors with respect to chorismate in the mutase reaction and with respect to chorismate in the mutase reaction and with respect to prephenate in the dehydrogenase reaction. The similarity of the dissociation constants for the interaction of these compounds with the free enzyme, as determined from the mutase and dehydrogenase reactions, indicates that the reaction of these inhibitors at a single site prevents the binding of both chorismate and prephenate. However, not all the groups on the enzyme, which are responsible for the binding of these two substrates, can be identical. At lower concentrations, citrate or malonate prevents reaction of the enzyme with prephenate, but not with chorismate. Nevertheless, the combining sites for chorismate and prephenate are in such close proximity that the diethyl derivative of malonate prevents the binding of both substrates. The results lead to the proposal that the sites at which chorismate and prephenate react on hydroxyphenylpyruvate synthase share common features and can be considered to overlap.     

A single cyclohexadienyl dehydratase specifies the prephenate dehydratase and arogenate dehydratase components of one of two independent pathways to L-phenylalanine in Erwinia herbicola

Dual biosynthetic pathways diverge from prephenate to L-phenylalanine in Erwinia herbicola, the unique intermediates of these pathways being phenylpyruvate and L-arogenate. After separation from the bifunctional P-protein (one component of which has prephenate dehydratase activity), the remaining prephenate dehydratase activity could not be separated from arogenate dehydratase activity throughout fractionation steps yielding a purification of more than 1200-fold. The ratio of activities was constant after removal of the P-protein, and the two dehydratase activities were stable during purification. Hence, the enzyme is a cyclohexadienyl dehydratase. The native enzyme has a molecular mass of 73 kDa and is a tetramer made up of identical 18-kDa subunits. Km values of 0.17 mM and 0.09 mM were calculated for prephenate and L-arogenate, respectively. L-Arogenate inhibited prephenate dehydratase competitively with respect to prephenate, whereas prephenate inhibited arogenate dehydratase competitively with respect to L-arogenate. Thus, the enzyme has a common catalytic site for utilization of prephenate or L-arogenate as alternative substrates. This is the first characterization of a purified monofunctional cyclohexadienyl dehydratase.     

Three Different Classes of Aminotransferases Evolved Prephenate Aminotransferase Functionality in Arogenate-competent Microorganisms

The aromatic amino acids phenylalanine and tyrosine represent essential sources of high value natural aromatic compounds for human health and industry. Depending on the organism, alternative routes exist for their synthesis. Phenylalanine and tyrosine are synthesized either via phenylpyruvate/4-hydroxyphenylpyruvate or via arogenate. In arogenate-competent microorganisms, an aminotransferase is required for the transamination of prephenate into arogenate, but the identity of the genes is still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1β in tested α- and β-proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the β-proteobacterium Nitrosomonas europaea. Recombinant PAT enzymes exhibit high activity toward prephenate, indicating that the corresponding genes encode bona fide PAT. PAT functionality was acquired without other modification of substrate specificity and is not a general catalytic property of the three classes of aminotransferases.     

Clues from Xanthomonas campestris about the evolution of aromatic biosynthesis and its regulation

The recent placement of major Gram-negative prokaryotes (Superfamily B) on a phylogenetic tree (including, e.g., lineages leading to Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus) has allowed initial insights into the evolution of the biochemical pathway for aromatic amino acid biosynthesis and its regulation to be obtained. Within this prokaryote grouping, Xanthomonas campestris ATCC 12612 (a representative of the Group V pseudomonads) has played a key role in facilitating deductions about the major evolutionary events that shaped the character of aromatic biosynthesis within this grouping. X. campestris is like P. aeruginosa (and unlike E. coli) in its possession of dual flow routes to both L-phenylalanine and L-tyrosine from prephenate. Like all other members of Superfamily B, X. campestris possesses a bifunctional P-protein bearing the activities of both chorismate mutase and prephenate dehydratase. We have found an unregulated arogenate dehydratase similar to that of P. aeruginosa in X. campestris. We separated the two tyrosine-branch dehydrogenase activities (prephenate dehydrogenase and arogenate dehydrogenase); this marks the first time this has been accomplished in an organism in which these two activities coexist. Superfamily B organisms possess 3-deoxy-D-arabino-heptulosonate 7-P (DAHP) synthase as three isozymes (e.g., in E. coli), as two isozymes (e.g., in P. aeruginosa), or as one enzyme (in X. campestris). The two-isozyme system has been deduced to correspond to the ancestral state of Superfamily B. Thus, E. coli has gained an isozyme, whereas X. campestris has lost one. We conclude that the single, chorismate-sensitive DAHP synthase enzyme of X. campestris is evolutionarily related to the tryptophan-sensitive DAHP synthase present throughout the rest of Superfamily B. In X. campestris, arogenate dehydrogenase, prephenate dehydrogenase, the P-protein, chorismate mutase-F, anthranilate synthase, and DAHP synthase are all allosteric proteins; we compared their regulatory properties with those of enzymes of other Superfamily B members with respect to the evolution of regulatory properties. The network of sequentially operating circuits of allosteric control that exists for feedback regulation of overall carbon flow through the aromatic pathway in X. campestris is thus far unique in nature.     

L-tyrosine regulation and biosynthesis via arogenate dehydrogenase in suspension-cultured cells of Nicotiana silvestris Speg. et Comes

The biosynthetic route to L-tyrosine was identified in isogenic suspension-cultured cells of N. silvestris. Arogenate (NADP⁺) dehydrogenase, the essential enzyme responsible for the conversion of L-arogenato L-tyrosine, was readily observed in crude extracts. In contrast, prephenate dehydrogenase (EC 1.3.1.13) activity with either NAD⁺ or NADP⁺ was absent altogether. Therefore, it seems likely that this tobacco species utilizes the arogenate pathway as the exclusive metabolic route to L-tyrosine. L-Tyrosine (but not L-phenylalanine) was a very effective endproduct inhibitor of arogenate dehydrogenase. In addition, analogs of L-tyrosine (m-fluoro-DL-tyrosine [MFT], D-tyrosine and N-acetyl-DL-tyrosine), but not of L-phenylalanine (o-fluoro-DL-phenylalanine and p-fluoro-DL-phenylalanine), were able to cause inhibition of arogenate dehydrogenase. The potent antimetabolite of L-tryptophan, 6-fluoro-DL-tryptophan, had no effect upon arogenate dehydrogenase activity. Of the compounds tested, MFT was actually more effective as an inhibitor of arogenate dehydrogenase than was L-tyrosine. Since MFT was found to be a potent antimetabolite inhibitor of growth in N. silvestris and since inhibition was specifically and effectively reversed by L-tyrosine, arogenate dehydrogenase is an outstanding candidate as the in vivo target of analog action. Although chorismate mutase (EC 5.4.99.5) cannot be the prime target of MFT action, MFT can mimick L-tyrosine in partially inhibiting this enzyme activity. The activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was insensitive to L-phenylalanine or L-tyrosine. The overall features of this system indicate that MFT should be a very effective analog mimick for selection of feedback-insensitive regulatory mutants L-tyrosine biosynthesis.Abbreviations DAHP synthase 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase - 6FT 6-fluoro-DL-tryptophan - MFT m-fluoro-DL-tyrosine - OFP o-fluoro-DL-phenylalanine - PFP p-fluoro-DL-phenylalanine     

Clues fromXanthomonas campestris about the evolution of aromatic biosynthesis and its regulation

Summary The recent placement of major Gramnegative prokaryotes (Superfamily B) on a phylogenetic tree (including, e.g., lineages leading toEscherichia coli, Pseudomonas aeruginosa, andAcinetobacter calcoaceticus) has allowed initial insights into the evolution of the biochemical pathway for aromatic amino acid biosynthesis and its regulation to be obtained. Within this prokaryote grouping,Xanthomonas campestris ATCC 12612 (a representative of the Group V pseudomonads) has played a key role in facilitating deductions about the major evolutionary events that shaped the character of aromatic biosynthesis within this grouping.X. campestris is likeP. aeruginosa (and unlikeE. coli) in its possession of dual flow routes to bothl-phenylalanine andl-tyrosine from prephenate. Like all other members of Superfamily B,X. campestris possesses a bifunctional P-protein bearing the activities of both chorismate mutase and prephenate dehydratase. We have found an unregulated arogenate dehydratase similar to that ofP. aeruginosa inX. campestris. We separated the two tyrosine-branch dehydrogenase activities (prephenate dehydrogenase and arogenate dehydrogenase); this marks the first time this has been accomplished in an organism in which these two activities coexist. Superfamily B organisms possess 3-deoxy-d-arabino-heptulosonate 7-P (DAHP) synthase as three isozymes (e.g., inE. coli), as two isozymes (e.g., inP. aeruginosa), or as one enzyme (inX. campestris). The two-isozyme system has been deduced to correspond to the ancestral state of Superfamily B. Thus,E. coli has gained an isozyme, whereasX. campestris has lost one. We conclude that the single, chorismate-sensitive DAHP synthase enzyme ofX. campestris is evolutionarily related to the tryptophan-sensitive DAHP synthase present throughout the rest of Superfamily B. InX. campestris, arogenate dehydrogenase, prephenate dehydrogenase, the P-protein, chorismate mutase-F, anthranilate synthase, and DAHP synthase are all allosteric proteins; we compared their regulatory properties with those of enzymes of other Superfamily B members with respect to the evolution of regulatory properties. The network of sequentially operating circuits of allosteric control that exists for feedback regulation of overall carbon flow through the aromatic pathway inX. campestris is thus far unique in nature.     

Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway

The aromatic amino acid Phe is required for protein synthesis and serves as the precursor of abundant phenylpropanoid plant natural products. While Phe is synthesized from prephenate exclusively via a phenylpyruvate intermediate in model microbes, the alternative pathway via arogenate is predominant in plant Phe biosynthesis. However, the molecular and biochemical evolution of the plant arogenate pathway is currently unknown. Here, we conducted phylogenetically informed biochemical characterization of prephenate aminotransferases (PPA-ATs) that belong to class-Ib aspartate aminotransferases (AspAT Ibs) and catalyze the first committed step of the arogenate pathway in plants. Plant PPA-ATs and succeeding arogenate dehydratases (ADTs) were found to be most closely related to homologs from Chlorobi/Bacteroidetes bacteria. The Chlorobium tepidum PPA-AT and ADT homologs indeed efficiently converted prephenate and arogenate into arogenate and Phe, respectively. A subset of AspAT Ib enzymes exhibiting PPA-AT activity was further identified from both Plantae and prokaryotes and, together with site-directed mutagenesis, showed that Thr-84 and Lys-169 play key roles in specific recognition of dicarboxylic keto (prephenate) and amino (aspartate) acid substrates. The results suggest that, along with ADT, a gene encoding prephenate-specific PPA-AT was transferred from a Chlorobi/Bacteroidetes ancestor to a eukaryotic ancestor of Plantae, allowing efficient Phe and phenylpropanoid production via arogenate in plants today.     

Evolutionary implications of features of aromatic amino acid biosynthesis in the genus Acinetobacter

Key enzymes of aromatic amino acid biosynthesis were examined in the genus Acinetobacter. Members of this genus belong to a suprafamilial assemblage of Gram-negative bacteria (denoted Superfamily B) for which a phylogenetic tree based upon oligonucleotide cataloging of 16S rRNA exists. Since the Acinetobacter lineage diverged at an early evolutionary time from other lineages within Superfamily B, an examination of aromatic biosynthesis in members of this genus has supplied improtant clues for the deduction of major evolutionary events leading to the contemporary aromatic pathways that now exist within Superfamily B. Together with Escherichia coli, Pseudomonas aeruginosa and Xanthomonas campestris, four well-spaced lineages have now been studied in comprehensive detail with respect to comparative enzymological features of aromatic amino acid biosynthesis. A. calcoaceticus and A. lwoffii both possess two chorismate mutase isozymes: one a monofunctional isozyme (chorismate mutase-F), and the other (chorismate mutase-P) a component of a bifunctional P-protein (chorismate mutase-prephenate dehydratase). While both P-protein activities were feedback inhibited by l-phenylalanine, the chorismate mutase-P activity was additionally inhibited by prephenate. Likewise, chorismate mutase-F was product inhibited by prephenate. Two isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase were detected. The major isozyme (>95%) was sensitive to feedback inhibition by l-tyrosine, whereas the minor isozyme was apparently insensitive to allosteric control. Prephenate dehydrogenase and arogenate dehydrogenase activities were both detected, but could not be chromatographically resolved. Available evidence favors the existence of a single dehydrogenase enzyme, exhibiting substrate ambiguity for prephenate andl-arogenate. Dehydrogenase activity with either of the latter substrates was specific for NADP⁺, NAD⁺ being ineffective. Consideration of the phylogeny of Superfamily-B organisms suggests that the stem ancestor of the Superfamily possessed a single dehydrogenase enzyme having ambiguity for both substrate and pyridine nucleotide cofactor. Since all other members of Superfamily B have NAD⁺-specific dehydrogenases, specialization for NADP⁺ must have occurred following the point of Acinetobacter divergence, leading to the dichotomy seen in present-day Superfamily-B organisms.     

Amino Acid Composition of Elm Seeds

In the biosynthetic pathway of aromatic amino acids of Brevibacterium flavum, ratios of each biosynthetic flow at the chorismate branch point were calculated from the reaction velocities of anthranilate synthetase for tryptophan and chorismate mutase for phenylalanine and tyrosine at steady state concentrations of chorismate. When these aromatic amino acids were absent, the ratio was 61, showing an extremely preferential synthesis of tryptophan. The presence of tryptophan at 0.01 mM decreased the ratio to 0.07, showing a diversion of the preferential synthesis to phenylalanine and tyrosine. Complete recovery by glutamate of the ability to synthesize the Millon-positive substance in dialyzed cell extracts confirmed that tyrosine was synthesized via pretyrosine in this organism. Partially purified prephenate aminotransferase, the first enzyme in the tyrosine-specific branch, had a pH optimum of 8.0 and Km’s of 0.45 and 22 mM for prephenate and glutamate, respectively, and its activity was increased 15-fold by pyridoxal-5-phosphate. Neither its activity nor its synthesis was affected at all by the presence of the end product tyrosine or other aromatic amino acids. The ratio of each biosynthetic flow for tyrosine and phenylalanine at the prephenate branch point was calculated from the kinetic equations of prephenate aminotransferase and prephenate dehydratase, the first enzyme in the phenylalanine-specific branch. It showed that tyrosine was synthesized in preference to phenylalanine when phenylalanine and tyrosine were absent. Furthermore, this preferential synthesis was diverted to a balanced synthesis of phenylalanine and tyrosine through activation of prephenate dehydratase by the tyrosine thus synthesized. The feedback inhibition of prephenate dehydratase by phenylalanine was proposed to play a role in maintaining a balanced synthesis when supply of prephenate was decreased by feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP*) synthetase, the common key enzyme. Overproduction of the end products in various regulatory mutants was also explained by these results.     

Enzyme Alterations in Tyrosine and Phenylalanine Auxotrophs of Salmonella typhimurium

The enzyme activities specified by the tyrA and pheA genes were studied in wildtype strain Salmonella typhimurium and in phenylalanine and tyrosine auxotrophs. As in Aerobacter aerogenes and Escherichia coli, the wild-type enzymes of Salmonella catalyze two consecutive reactions: chorismate --> prephenate --> 4-hydroxy-phenylpyruvate (tyrA), and chorismate --> prephenate --> phenylpyruvate (pheA). A group of tyrA mutants capable of interallelic complementation had altered enzymes which retained chorismate mutase T activity but lacked prephenate dehydrogenase. Similarly, pheA mutants (in which interallelic complementation does not occur) had one group with altered enzymes which retained chorismate mutase P but lacked prephenate dehydratase. Tyrosine and phenylalanine auxotrophs outside of these categories showed loss of both activities of their respective bifunctional enzyme. TyrA mutants which had mutase T were considerably derepressed in this activity by tyrosine starvation and consequently excreted prephenate. A new and specific procedure was developed for assaying prephenate dehydrogenase activity.     

Enzymological basis for herbicidal action of glyphosate

The effects of 1 millimolar glyphosate (N-[phosphonomethyl]glycine) upon the activities of enzymes of aromatic amino acid biosynthesis, partially purified by ion-exchange chromatography from mung bean seedings (Vigna radiata [L.] Wilczek), were examined. Multiple isozyme species of shikimate dehydrogenase, chorismate mutase, and aromatic aminotransferase were separated, and these were all insensitive to inhibition by glyphosate. The activities of prephenate dehydrogenase and arogenate dehydrogenase were also not sensitive to inhibition. Two molecular species of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase were resolved, one stimulated several-fold by Mn²⁺ (DAHP synthase-Mn), and the other absolutely dependent upon the presence of Co²⁺ for activity (DAHP synthase-Co). Whereas DAHP synthase-Mn was invulnerable to glyphosate, greater than 95% inhibition of DAHP synthase-Co was found in the presence of glyphosate. Since Co²⁺ is a V_max activator with respect to both substrates, glyphosate cannot act simply by Co²⁺ chelation because inhibition is competitive with respect to erythrose-4-phosphate. The accumulation of shikimate found in glyphosate-treated seedlings is consistent with in vivo inhibition of both 5-enolpyruvylshikimic acid 3-phosphate synthase and one of the two DAHP synthase isozymes. Aromatic amino acids, singly or in combination, only showed a trend towards reversal of growth inhibition in 7-day seedlings of mung bean. The possibilities are raised that glyphosate may act at multiple enzyme targets in a given organism or that different plants may vary in the identity of the prime enzyme target.     

A single cyclohexadienyl dehydrogenase specifies the prephenate dehydrogenase and arogenate dehydrogenase components of the dual pathways to L-tyrosine in Pseudomonas aeruginosa

Dual biosynthetic pathways diverge from prephenate to L-tyrosine in Pseudomonas aeruginosa, with 4-hydroxyphenylpyruvate and L-arogenate being the unique intermediates of these pathways. Prephenate dehydrogenase and arogenate dehydrogenase activities could not be separated throughout fractionation steps yielding a purification of more than 200-fold, and the ratio of activities was constant throughout purification. Thus, the enzyme is a cyclohexadienyl dehydrogenase. The native enzyme has a molecular weight of 150,000 and is a hexamer made up of identical 25,500 subunits. The enzyme is specific for NAD+ as an electron acceptor, and identical Km values of 0.25 mM were obtained for NAD+, regardless of whether activity was assayed as prephenate dehydrogenase or as arogenate dehydrogenase. Km values of 0.07 mM and 0.17 mM were calculated for prephenate and L-arogenate, respectively. Inhibition by L-tyrosine was noncompetitive with respect to NAD+, but was strictly competitive with respect to either prephenate or L-arogenate. With cyclohexadiene as variable substrate, similar Ki values for L-tyrosine of 0.06 mM (prephenate) and 0.05 mM (L-arogenate) were obtained. With NAD+ as the variable substrate, similar Ki values for L-tyrosine of 0.26 mM (prephenate) and 0.28 mM (L-arogenate), respectively, were calculated. This is the first characterization of a purified, monofunctional cyclohexadienyl dehydrogenase.     

The aromatic amino acid pathway branches at L-arogenate in Euglena gracilis.

The recently characterized amino acid L-arogenate (Zamir et al., J. Am. Chem. Soc. 102:4499-4504, 1980) may be a precursor of either L-phenylalanine or L-tyrosine in nature. Euglena gracilis is the first example of an organism that uses L-arogenate as the sole precursor of both L-tyrosine and L-phenylalanine, thereby creating a pathway in which L-arogenate rather than prephenate becomes the metabolic branch point. E. gracilis ATCC 12796 was cultured in the light under myxotrophic conditions and harvested in late exponential phase before extract preparation for enzymological assays. Arogenate dehydrogenase was dependent upon nicotinamide adenine dinucleotide phosphate for activity. L-Tyrosine inhibited activity effectively with kinetics that were competitive with respect to L-arogenate and noncompetitive with respect to nicotinamide adenine dinucleotide phosphate. The possible inhibition of arogenate dehydratase by L-phenylalanine has not yet been determined. Beyond the latter uncertainty, the overall regulation of aromatic biosynthesis was studied through the characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and chorismate mutase. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase was subject to noncompetitive inhibition by L-tyrosine with respect to either of the two substrates. Chorismate mutase was feedback inhibited with equal effectiveness by either L-tyrosine or L-phenylalanine. L-Tryptophan activated activity of chorismate mutase, a pH-dependent effect in which increased activation was dramatic above pH 7.8 L-Arogenate did not affect activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase or of chorismate mutase. Four species of prephenate aminotransferase activity were separated after ion-exchange chromatography. One aminotransferase exhibited a narrow range of substrate specificity, recognizing only the combination of L-glutamate with prephenate, phenylpyruvate, or 4-hydroxyphenylpyruvate. Possible natural relationships between Euglena spp. and fungi previously considered in the literature are discussed in terms of data currently available to define enzymological variation in the shikimate pathway.     

Complementation of the pha2 yeast mutant suggests functional differences for arogenate dehydratases from Arabidopsis thaliana

The final steps of phenylalanine (Phe) biosynthesis in bacteria, fungi and plants can occur via phenylpyruvate or arogenate intermediates. These routes are determined by the presence of prephenate dehydratase (PDT, EC4.2.1.51), which forms phenylpyruvate from prephenate, or arogenate dehydratase (ADT, EC4.2.1.91), which forms phenylalanine directly from arogenate. We compared sequences from select yeast species to those of Arabidopsis thaliana. The in silico analysis showed that plant ADTs and yeast PDTs share many common features allowing them to act as dehydratase/decarboxylases. However, plant and yeast sequences clearly group independently conferring distinct substrate specificities. Complementation of the Saccharomyces cerevisiae pha2 mutant, which lacks PDT activity and cannot grow in the absence of exogenous Phe, was used to test the PDT activity of A. thaliana ADTs in vivo. Previous biochemical characterization showed that all six AtADTs had high catalytic activity with arogenate as a substrate, while AtADT1, AtADT2 and AtADT6 also had limited activity with prephenate. Consistent with these results, the complementation test showed AtADT2 readily recovered the pha2 phenotype after ～6 days growth at 30 °C, while AtADT1 required ～13 days to show visible growth. By contrast, AtADT6 (lowest PDT activity) and AtADT3-5 (no PDT activity) were unable to recover the phenotype. These results suggest that only AtADT1 and AtADT2, but not the other four ADTs from Arabidopsis, have functional PDT activity in vivo, showing that there are two functional distinct groups. We hypothesize that plant ADTs have evolved to use the arogenate route for Phe synthesis while keeping some residual PDT activity.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 1. Kinetic characterization of the dehydrogenase reaction by use of alternative substrates

The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase-prephenate dehydrogenase, has been isolated from extracts of a plasmid-containing strain of Escherichia coli K12 and purified to homogeneity by a modified procedure that involves chromatography on both Matrex Blue A and Sepharose-AMP. Detailed studies of the dehydrogenase reaction have been undertaken with analogues of prephenate that act as substrates. The analogues, which included two of the four possible diastereoisomers of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate (deoxodihydroprephenate) as well as D- and L-arogenate, were synthesized chemically. As judged by their V/K values, all analogues were poorer substrates than prephenate. The order of their effectiveness as substrates is prephenate greater than one isomer of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate greater than L-arogenate greater than other isomer of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate greater than D-arogenate. Thus the dehydrogenase activity is dependent on the degree and position of unsaturation in the ring structure of prephenate as well as on the type of substitution on the pyruvyl side chain. With prephenate as a substrate, the reaction is irreversible because it involves oxidative decarboxylation. By contrast, 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate undergoes only a simple oxidation, and thus, with this substrate, the reaction is reversible. Steady-state velocity data, obtained by varying substrates over a range of higher concentrations, suggest that the dehydrogenase reaction conforms to a rapid equilibrium, random mechanism with 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate as a substrate in the forward reaction or with the corresponding ketone derivative as a substrate in the reverse direction. The initial velocity patterns obtained by varying prephenate or 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate over a range of lower concentrations, at different fixed concentrations of NAD, were nonlinear and consistent with a unique model that is described by a velocity equation which is the ratio of quadratic polynomials. An equilibrium constant of 1.4 x 10(-7) M for the reaction in the presence of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate indicates that the equilibrium lies very much in favor of ketone production.     

Phenylalanine biosynthesis in Arabidopsis thaliana. Identification and characterization of arogenate dehydratases

There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had k(cat)/Km values of 1050, 7650, and 1560 M(-1) S(-1) for arogenate versus 38, 240, and 16 M(-1) S(-1) for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had k(cat)/Km values of 1140, 490, and 620 M(-1) S(-1), with prephenate not serving as a substrate unless excess recombinant protein (>150 microg/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1-ADT6.     

Chorismate mutase-prephenate dehydratase from Escherichia coli: active sites of a bifunctional enzyme.

The relationship between the active sites of the bifunctional enzyme chorismate mutase-prephenate dehydratase has been examined. Steady-state kinetic investigations of the reactions with chorismate or prephenate as substrate and studies of the overall conversion of chorismate to phenylpyruvate indicate that there are two distinct active sites. One site is responsible for the mutase activity and the other for the dehydratase activity. Studies of the overall reaction using radioactive chorismate show that prephenate, which is formed from chorismate, dissociates from the mutase site and equilibrates with the bulk medium before combining at the dehydratase site. No evidence was obtained for direct channeling of prephenate from one site to the other, or for any strong interaction between the sites.     

Crystal structure of prephenate dehydrogenase from Aquifex aeolicus. Insights into the catalytic mechanism

The enzyme prephenate dehydrogenase catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate for the biosynthesis of tyrosine. Prephenate dehydrogenases exist as either monofunctional or bifunctional enzymes. The bifunctional enzymes are diverse, since the prephenate dehydrogenase domain is associated with other enzymes, such as chorismate mutase and 3-phosphoskimate 1-carboxyvinyltransferase. We report the first crystal structure of a monofunctional prephenate dehydrogenase enzyme from the hyper-thermophile Aquifex aeolicus in complex with NAD+. This protein consists of two structural domains, a modified nucleotide-binding domain and a novel helical prephenate binding domain. The active site of prephenate dehydrogenase is formed at the domain interface and is shared between the subunits of the dimer. We infer from the structure that access to the active site is regulated via a gated mechanism, which is modulated by an ionic network involving a conserved arginine, Arg250. In addition, the crystal structure reveals for the first time the positions of a number of key catalytic residues and the identity of other active site residues that may participate in the reaction mechanism; these residues include Ser126 and Lys246 and the catalytic histidine, His147. Analysis of the structure further reveals that two secondary structure elements, beta3 and beta7, are missing in the prephenate dehydrogenase domain of the bifunctional chorismate mutase-prephenate dehydrogenase enzymes. This observation suggests that the two functional domains of chorismate mutase-prephenate dehydrogenase are interdependent and explains why these domains cannot be separated.