Hidden overflow pathway to L-phenylalanine in Pseudomonas aeruginosa

Pseudomonas aeruginosa is representative of a large group of pseudomonad bacteria that possess coexisting alternative pathways to L-phenylalanine (as well as to L-tyrosine). These multiple flow routes to aromatic end products apparently account for the inordinate resistance of P. aeruginosa to end product analogs. Manipulation of carbon source nutrition produced a physiological state of sensitivity to p-fluorophenylalanine and m-fluorophenylalanine, each a specific antimetabolite of L-phenylalanine. Analog-resistant mutants obtained fell into two classes. One type lacked feedback sensitivity of prephenate dehydratase and was the most dramatic excretor of L-phenylalanine. The presence of L-tyrosine curbed phenylalanine excretion to one-third, a finding explained by potent early-pathway regulation of 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase-Tyr (a DAHP synthase subject to allosteric inhibition by L-tyrosine). The second class of regulatory mutants possessed a completely feedback-resistant DAHP synthase-Tyr, the major species (greater than 90%) of two isozymes. Deregulation of DAHP synthase-Tyr resulted in the escape of most chorismate molecules produced into an unregulated overflow route consisting of chorismate mutase (monofunctional), prephenate aminotransferase, and arogenate dehydratase. In the wild type the operation of the overflow pathway is restrained by factors that restrict early-pathway flux. These factors include the highly potent feedback control of DAHP synthase isozymes by end products as well as the strikingly variable abilities of different carbon source nutrients to supply the aromatic pathway with beginning substrates. Even in the wild type, where all allosteric regulation in intact, some phenylalanine overflow was found on glucose-based medium, but not on fructose-based medium. This carbon source-dependent difference was much more exaggerated in each class of regulatory mutants.     

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.     

A Bifunctional Enzyme in Pseudomonas aeruginosa: a New Pattern in the Organization of Enzymes Concerned with Phenylalanine and Tyrosine Biosynthesis

Two isozymes of chorismate mutase (CA mutase(1) and CA mutase(2)) and two isozymes of prephenate dehydratase (PPA dehydratase(1) and PPA dehydratase(2)) have been found in Pseudomonas aeruginosa. The activities CA mutase(2)-PPA dehydratase(2) catalyzing phenylalanine biosynthesis have been purified almost 40-fold and were found to be associated as a bifunctional enzyme or an enzyme complex. The enzymes specific for tyrosine biosynthesis did not appear to manifest such physical association. Thus, the organization of enzymes concerned with phenylalanine and tyrosine biosynthesis in P. aeruginosa is unique and is unlike most other organisms. Single site mutants have been isolated which have lost both CA mutase(2)-PPA dehydratase(2) activities resulting in a requirement for phenylalanine for growth. Single site revertants of these mutants regained both these activities simultaneously and were able to grow on minimal medium. A mutant, r(6), was also isolated which had normal CA mutase(2) but lacked PPA dehydratase(2) activity.     

Enzymological basis for growth inhibition by L-phenylalanine in the cyanobacterium Synechocystis sp. 29108

The pattern of allosteric control in the biosynthetic pathway for aromatic amino acids provides a basis to explain vulnerability to growth inhibition by l-phenylalanine (0.2 mM or greater) in the unicellular cyanobacterium Synechocystis sp. 29108. We attribute growth inhibition to the hypersensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase to feedback inhibition by l-phenylalanine. Hyperregulation of this initial enzyme of aromatic biosynthesis depletes the supply of precursors needed for biosynthesis of l-tyrosine and l-tryptophan. Consistent with this mechanism is the total reversal of phenylalanine inhibition by a combination of tyrosine and tryptophan. Inhibited cultures also contained decreased levels of phycocyanin pigments, a characteristic previously correlated with amino acid starvation in cyanobacteria. l-Phenylalanine is a potent noncompetitive inhibitor (with both substrates) of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase, whereas l-tyrosine is a very weak inhibitor. Prephenate dehydratase also displays allosteric sensitivity to phenylalanine (inhibition) and to tyrosine (activation). Both 2-fluoro and 4-fluoro derivatives of phenylalanine were potent analog antimetabolites, and these were used in addition to l-phenylalanine as selective agents for resistant mutants. Mutants were isolated which excreted both phenylalanine and tyrosine, the consequence of an altered 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase no longer sensitive to feedback inhibition. Simultaneous insensitivity to l-tyrosine suggests that l-tyrosine acts as a weak analog mimic of l-phenylalanine at a common binding site. Prephenate dehydratase in the regulatory mutants was unaltered. Surprisingly, in view of the lack of regulation in the tyrosine branchlet of the pathway, such mutants excrete more phenylalanine than tyrosine, indicating that l-tyrosine activation dominates l-phenylalanine inhibition of prephenate dehydratase in vivo. In mutant Phe r19 the loss in allosteric sensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase was accompanied by a threefold increase in specific activity. This could suggest that existence of a modest degree of repression control (autogenous) over 3-deoxy-d-arabinoheptulosonate synthase, although other explanations are possible. Specific activities of chorismate mutase, prephenate dehydratase, shikimate/nicotinamide adenine dinucleotide phosphate dehydrogenase, and arogenate/nicotinamide adenine dinucleotide phosphate dehydrogenase in mutant Phe r19 were identical with those of the wild type.     

Biochemical diversity for biosynthesis of aromatic amino acids among the cyanobacteria.

We examined the enzymology and regulatory patterns of the aromatic amino acid pathway in 48 strains of cyanobacteria including representatives from each of the five major grouping. Extensive diversity was found in allosteric inhibition patterns of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, not only between the major groupings but also within several of the generic groupings. Unimetabolite inhibition by phenylalanine occurred in approximately half of the strains examined; in the other strains unimetabolite inhibition by tyrosine and cumulative, concerted, and additive patterns were found. The additive patterns suggest the presence of regulatory isozymes. Even though both arogenate and prephenate dehydrogenase activities were found in some strains, it seems clear that the arogenate pathway to tyrosine is a common trait that has been highly conserved among cyanobacteria. No arogenate dehydratase activities were found. In general, prephenate dehydratase activities were activated by tyrosine and inhibited by phenylalanine. Chorismate mutase, arogenate dehydrogenase, and shikimate dehydrogenase were nearly always unregulated. Most strains preferred NADP as the cofactor for the dehydrogenase activities. The diversity in the allosteric inhibition patterns for 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, cofactor specificities, and the presence or absence of prephenate dehydrogenase activity allowed the separation of subgroupings within several of the form genera, namely, Synechococcus, Synechocystis, Anabaena, Nostoc, and Calothrix.     

Phenylalanine biosynthesis and its relationship to accumulation of capsaicinoids during Capsicum chinense fruit development

Activities of phenylalanine (Phe) biosynthetic enzymes chorismate mutase (CM) and arogenate dehydratase (ADT) and of phenylalanine ammonia lyase [PAL, an enzyme that directs Phe towards capsaicinoid (CAP) synthesis] were analyzed during Capsicum chinense Jacq. (habanero pepper) fruit development. A maximum CM activity coincided with a maximum CAP accumulation. However, ADT exhibited two activity peaks, one during the early phase (10 - 17 days post-anthesis, DPA) and another during the late phase (35 - 37 DPA); only the latter coincided with CAP. Interestingly, PAL activity was inversely related to CAP accumulation; lower activities coincided with a maximum CAP content. These results suggest the operation of a control mechanism that coordinated Phe synthesis and its channeling towards CAP synthesis during the course of fruit development.     

Chorismate mutase:prephenate dehydratase from Acinetobacter calcoaceticus. Purification, properties and immunological cross-reactivity

The bifunctional P protein (chorismate mutase: prephenate dehydratase) from Acinetobacter calcoaceticus has been purified. It was homogeneous in polyacrylamide gels and was more than 95% pure on the basis of the immunostaining of purified P protein with the antibodies raised against the P protein. The native enzyme is a homodimer (Mr = 91,000) composed of 45-kDa subunits. A twofold increase in the native molecular mass of the P protein occurred in the presence of L-phenylalanine (inhibitor of both activities) or L-tyrosine (activator of the dehydratase activity) during gel filtration. Chorismate mutase activity followed Michaelis-Menten kinetics with a Km of 0.55 mM for chorismate. L-Phenylalanine was a relatively poor non-competitive inhibitor of the mutase activity. The chorismate mutase activity was also competitively inhibited by prephenate (reaction product). Substrate-saturation curves for the dehydratase activity were sigmoidal showing positive cooperativity among the prephenate-binding sites. L-Tyrosine activated prephenate dehydratase strongly but did not abolish positive cooperativity with respect to prephenate. L-Phenylalanine inhibited the dehydratase activity, and the substrate-saturation curves became increasingly sigmoidal as phenylalanine concentrations were increased with happ values changing from 2.0 (no phenylalanine) to 4.0 (0.08 mM L-phenylalanine). A sigmoidal inhibition curve of the dehydratase activity by L-phenylalanine gave Hill plots having a slope of -2.9. Higher ionic strength increased the dehydratase activity by reducing the positive cooperative binding of prephenate, and the sigmoidal substrate-saturation curves were changed to near-hyperbolic form. The happ values decreased with increase in ionic strength. Antibodies raised against the purified P protein showed cross-reactivity with the P proteins from near phylogenetic relatives of A. calcoaceticus. At a greater phylogenetic distance, cross-reaction was superior with P protein from Neisseria gonorrhoeae than with that from the more closely related Escherichia coli.     

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.     

Terminal phenylalanine and tyrosine biosynthesis of Microtetraspora glauca

The enzymes of the terminal steps of the phenylalanine and tyrosine biosynthesis were partially purified and characterized in Microtetraspora glauca, a spore-forming member of the order Actinomycetales. This bacterium relies exclusively on the phenylpyruvate route for phenylalanine synthesis, no arogenate dehydratase activity being found. Prephenate dehydratase is subject to feedback inhibition by phenylalanine, tyrosine and tryptophan, each acting as competitive inhibitor by increasing the Km of 72 microM for prephenate. Based on the results of gel chromatography on Sephadex G-200, the molecular mass of about 110,000 Da is not altered by any of the effectors. The enzyme is quite sensitive to inhibition by 4-hydroxymercuribenzoate. Microtetraspora glauca can utilize arogenate and 4-hydroxyphenylpyruvate as intermediates in tyrosine biosynthesis. Prephenate and arogenate dehydrogenase activities copurifying from ion exchange columns with coincident profiles were detected. From gel-filtration columns the two activities eluted at an identical molecular-mass position of about 68,000 Da. The existence of a single protein exhibiting substrate ambiguity is consistent with the findings, that both dehydrogenases have similar chromatographic properties, exhibit cofactor requirement for NAD and are inhibited to the same extent by tyrosine and 4-hydroxymercuribenzoate.     

Two biosynthetic pathways for aromatic amino acids in the archaeon Methanococcus maripaludis

Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon. Aromatic amino acids (AroAAs) are biosynthesized in this autotroph either by the de novo pathway, with chorismate as an intermediate, or by the incorporation of exogenous aryl acids via indolepyruvate oxidoreductase (IOR). In order to evaluate the roles of these pathways, the gene that encodes the third step in the de novo pathway, 3-dehydroquinate dehydratase (DHQ), was deleted. This mutant required all three AroAAs for growth, and no DHQ activity was detectible in cell extracts, compared to 6.0 +/- 0.2 mU mg(-1) in the wild-type extract. The growth requirement for the AroAAs could be fulfilled by the corresponding aryl acids phenylacetate, indoleacetate, and p-hydroxyphenylacetate. The specific incorporation of phenylacetate into phenylalanine by the IOR pathway was demonstrated in vivo by labeling with [1-(13)C]phenylacetate. M. maripaludis has two IOR homologs. A deletion mutant for one of these homologs contained 76, 74, and 42% lower activity for phenylpyruvate, p-hydoxyphenylpyruvate, and indolepyruvate oxidation, respectively, than the wild type. Growth of this mutant in minimal medium was inhibited by the aryl acids, but the AroAAs partially restored growth. Genetic complementation of the IOR mutant also restored much of the wild-type phenotype. Thus, aryl acids appear to regulate the expression or activity of the de novo pathway. The aryl acids did not significantly inhibit the activity of the biosynthetic enzymes chorismate mutase, prephenate dehydratase, and prephenate dehydrogenase in cell extracts, so the inhibition of growth was probably not due to an effect on these enzymes.     

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.     

Regulation of enzyme synthesis in the aromatic amino acid pathway of Bacillus subtilus

The control of the synthesis of certain key enzymes of aromatic amino acid biosynthesis was studied. Tyrosine represses the first enzyme of the 3-deoxy-d-arabino heptulosonic acid 7-phosphate pathway, DAHP synthetase, as well as shikimate kinase and chorismate mutase about fivefold in cultures grown under conditions limiting the synthesis of the aromatic amino acids. A mixture of tyrosine and phenylalanine represses twofold further. Tryptophan does not appear to be involved in the control of these enzymes. The specific activity of at least one early enzyme, dehydroquinase, remains essentially constant under a variety of nutritional supplementations. Two enzymes in the terminal branches are repressed by the amino acids they help to synthesize: prephenate dehydrogenase can be repressed fourfold by tyrosine, and anthranilate synthetase can be repressed over 200-fold by tryptophan. There is no evidence that phenylalanine represses prephenate dehydratase. Regulatory mutants have been isolated in which various enzymes of the pathway are no longer repressible. One class is derepressed for several of the prechorismate enzymes, as well as chorismate mutase and prephenate dehydrogenase. In another mutant, several enzymes of tryptophan biosynthesis are no longer repressible. Thus, the rate of synthesis of enzymes at every stage of the pathway is under control of various aromatic amino acids. Tyrosine and phenylalanine control the synthesis of enzymes involved in the synthesis of the three aromatic amino acids. Each terminal branch is under the control of its end product.     

Cyclohexadienyl dehydratase from Pseudomonas aeruginosa. Molecular cloning of the gene and characterization of the gene product.

The gene encoding cyclohexadienyl dehydratase (denoted pheC) was cloned from Pseudomonas aeruginosa by functional complementation of a pheA auxotroph of Escherichia coli. The gene was highly expressed in E. coli due to the use of the high-copy number vector pUC18. The P. aeruginosa cyclohexadienyl dehydratase expressed in E. coli was purified to electrophoretic homogeneity. The latter enzyme exhibited identical physical and biochemical properties as those obtained for cyclohexadienyl dehydratase purified from P. aeruginosa. The activity ratios of prephenate dehydratase to arogenate dehydratase remained constant (about 3.3-fold) throughout purification, thus demonstrating a single protein having broad substrate specificity. The cyclohexadienyl dehydratase exhibited Km values of 0.42 mM for prephenate and 0.22 mM for L-arogenate, respectively. The pheC gene was 807 base pairs in length, encoding a protein with a calculated molecular mass of 30,480 daltons. This compares with a molecular mass value of 29.5 kDa determined for the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since the native molecular mass determined by gel filtration was 72 kDa, the enzyme probably is a homodimer. Comparison of the deduced amino acid sequence of pheC from P. aeruginosa with those of the prephenate dehydratases of Corynebacterium glutamicum, Bacillus subtilis, E. coli, and Pseudomonas stutzeri by standard pairwise alignments did not establish obvious homology. However, a more detailed analysis revealed a conserved motif (containing a threonine residue known to be essential for catalysis) that was shared by all of the dehydratase proteins.     

Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum.

The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed.     

Metabolic Engineering To Produce Tyrosine or Phenylalanine in a Tryptophan-Producing Corynebacterium glutamicum Strain

The aromatic amino acids are synthesized via a common biosynthetic pathway. A tryptophan-producing mutant of Corynebacterium glutamicum was genetically engineered to produce tyrosine or phenylalanine in abundance. To achieve this, three biosynthetic genes encoding the first enzyme in the common pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DS), and the branch-point enzymes chorismate mutase and prephenate dehydratase were individually cloned from regulatory mutants of C. glutamicum which have either of the corresponding enzymes desensitized to end product inhibition. These cloned genes were assembled one after another onto a multicopy vector of C. glutamicum to yield two recombinant plasmids. One plasmid, designated pKY1, contains the DS and chorismate mutase genes, and the other, designated pKF1, contains all three biosynthetic genes. The enzymes specified by both plasmids were simultaneously overexpressed approximately sevenfold relative to the chromosomally encoded enzymes in a C. glutamicum strain. When transformed with pKY1 or pKF1, tryptophan-producing C. glutamicum KY10865, with the ability to produce 18 g of tryptophan per liter, was altered to produce a large amount of tyrosine (26 g/liter) or phenylalanine (28 g/liter), respectively, because the accelerated carbon flow through the common pathway was redirected to tyrosine or phenylalanine.     

Enzymological features of aromatic amino acid biosynthesis reflect the phylogeny of mycoplasmas

Acholeplasma laidlawii possesses a biochemical pathway for tyrosine and phenylalanine biosynthesis, while Mycoplasma iowae and Mycoplasma gallinarum do not. The detection of 7-phospho-2-dehydro-3-deoxy-D-arabino-heptonate (DAHP) synthase (EC 4.1.2.15), dehydro-shikimate reductase (EC 1.1.1.25) and 3-enol-pyruvoylshikimate-5-phosphate synthase (EC 2.5.1.19) activities in cell-free extracts established the presence in A. laidlawii of a functional shikimate pathway. L-Phenylalanine synthesis occurs solely through the phenylpyruvate route via prephenate dehydratase (EC 4.2.1.51), no arogenate dehydratase activity being found. Although arogenate dehydrogenase was detected, L-tyrosine synthesis appears to occur mainly through the 4-hydroxyphenylpyruvate route, via prephenate dehydrogenase (EC 1.3.1.12), which utilized NAD+ as a preferred coenzyme substrate. L-Tyrosine was found to be the key regulatory molecule governing aromatic biosynthesis. DAHP synthase was feedback inhibited by L-tyrosine, but not by L-phenylalanine or L-tryptophan; L-tyrosine was a potent feedback inhibitor of prephenate dehydrogenase and an allosteric activator of prephenate dehydratase. Chorismate mutase (EC 5.4.99.5) was sensitive to product inhibition by prephenate. Prephenate dehydratase was feedback inhibited by L-phenylalanine. It was also activated by hydrophobic amino acids (L-valine, L-isoleucine and L-methionine), similar to results previously found in a number of other genera that share the Gram-positive line of phylogenetic descent. Aromatic-pathway-encoded cistrons present in saprophytic large-genome mycoplasmas may have been eliminated in the parasitic small-genome mycoplasmas.     

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.     

Synthesis of chiral arogenic acid via immobilized microbial proteins

Although l-(8S)-arogenate has been recognized as a potential precursor of l-phenylalanine or l-tyrosine biosynthesis for only a few years, it is widely distributed in nature. The biochemical formation of arogenate has involved its isolation from the culture supernatant of a mutant strain of Neurospora crassa, a lengthy procedure of 20-day duration. We now report an improved approach using immobilized crude enzyme extracts from a cyanobacterium. The starting materials, chorismic acid or prephenic acid, are readily available, and overall yields ranging from 40 to 60% are obtained. The whole procedure takes only 1 day. Crude, unfractionated enzyme extracts from Synechocystis sp. ATCC 29108 are immobilized on a phenoxyacetyl cellulose solid support. The hydrophobic binding of the extract proteins did not denature chorismate mutase or prephenate aminotransferase, the enzymes catalyzing the conversion of chorismate to prephenate and prephenate to arogenate, respectively. This microbial system was ideally suited for preparation of arogenate, since other enzyme activities which might compete for prephenate or chorismate as substrates, or which might further metabolize arogenate, were absent or inactive under the conditions used. In addition to the substrates prephenate or chorismate, pyridoxal-5′-phosphate (the coenzyme required for transamination), as well as leucine (amino donor for transamination of prephenate), was added. The reaction product, arogenate, was separated from the starting materials by preparative thin-layer chromatography.     

Pleiotropic physiological consequences of feedback‐insensitive phenylalanine biosynthesis in Arabidopsis thaliana

A large proportion of plant carbon flow passes through the shikimate pathway to phenylalanine, which serves as a precursor for numerous secondary metabolites. To identify new regulatory mechanisms affecting phenylalanine metabolism, we isolated Arabidopsis thaliana mutants that are resistant to the phytotoxic amino acid m‐tyrosine, a structural analog of phenylalanine. Map‐based cloning identified adt2‐1D, a dominant point mutation causing a predicted serine to alanine change in the regulatory domain of ADT2 (arogenate dehydratase 2). Relaxed feedback inhibition and increased expression of the mutant enzyme caused up to 160‐fold higher accumulation of free phenylalanine in rosette leaves, as well as altered accumulation of several other primary and secondary metabolites. In particular, abundance of 2‐phenylethylglucosinolate, which is normally almost undetectable in leaves of the A. thaliana Columbia‐0 accession, is increased more than 30‐fold. Other observed phenotypes of the adt2‐1D mutant include abnormal leaf development, resistance to 5‐methyltryptophan, reduced growth of the generalist lepidopteran herbivore Trichoplusia ni (cabbage looper) and increased salt tolerance.