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.

Evolution of L-phenylalanine biosynthesis in rRNA homology group I of Pseudomonas.

Group I pseudomonads exhibit diversity for L-phenylalanine biosynthesis that is a basis for separation of two subgroups. Subgroup Ib (fluorescent species such as Pseudomonas aeruginosa, P. fluorescens, or P. putida) possesses an unregulated overflow pathway to L-phenylalanine, together with a second, regulated pathway. Subgroup Ia (non-fluorescent species such as P. stutzeri, P. mendocina, or P. alcaligenes) possess only the regulated pathway to L-phenylalanine. Thus, subgroup Ia species lack an unregulated isozyme of chorismate mutase and arogenate dehydratase, enzymes which are thought to divert chorismate to L-phenylalanine under conditions of high carbon input into aromatic biosynthesis. A priori the overflow pathway could have been either lost in subgroup Ia or gained in subgroup Ib. Since Group V pseudomonads (mainly Xanthomonas) are known to branch off from the Group I lineage at a deeper phylogenetic level than the point of divergence for subgroups Ia and Ib, the presence of the overflow pathway in Group V pseudomonads reveals that the overflow pathway must have been lost in the evolution of subgroup Ia. All Group I species possess a bifunctional protein (P-protein) which catalyzes both chorismate mutase and prephenate dehydratase reactions. In subgroup Ia species this highly conserved protein must be the sole source of prephenate to be used for tyrosine biosynthesis. Thus, the channeling action of the P-protein whereby chorismate is committed towards L-phenylalanine formation can be negated by selective feedback inhibition exerted by L-phenylalanine upon the prephenate dehydratase component of the P-protein. Diversion of prephenate molecules under the latter conditions towards L-tyrosine comprises a channel-shuttle mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Pseudomonas aeruginosa possesses two novel regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

In Pseudomonas aeruginosa the initial enzyme of aromatic amino acid biosynthesis, 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, has been known to be subject to feedback inhibition by a metabolite in each of the three major pathway branchlets. Thus, an apparent balanced multieffector control is mediated by L-tyrosine, by L-tryptophan, and phenylpyruvate. We have now resolved DAHP synthase into two distinctive regulatory isozymes, herein denoted DAHP synthase-tyr (Mr = 137,000) and DAHP synthase-trp (Mr = 175,000). DAHP synthase-tyr comprises greater than 90% of the total activity. L-Tyrosine was found to be a potent effector, inhibiting competitively with respect to both phosphoenolpyruvate (Ki = 23 microM) and erythrose 4-phosphate (Ki = 23 microM). Phenylpyruvate was a less effective competitive inhibitor: phosphoenolpyruvate (Ki = 2.55 mM) and erythrose 4-phosphate (Ki = 1.35 mM). DAHP synthase-trp was found to be inhibited noncompetitively by L-tryptophan with respect to phosphoenolpyruvate (Ki = 40 microM) and competitively with respect to erythrose 4-phosphate (Ki = 5 microM). Chorismate was a relatively weak competitive inhibitor: phosphoenolpyruvate (Ki = 1.35 mM) and erythrose 4-phosphate (Ki = 2.25 mM). Thus, each isozyme is strongly inhibited by an amino acid end product and weakly inhibited by an intermediary metabolite.

A multispecific quintet of aromatic aminotransferases that overlap different biochemical pathways in Pseudomonas aeruginosa.

Pseudomonas aeruginosa possesses dual enzymatic sequences to both L-phenylalanine and L-tyrosine, a biosynthetic arrangement further complicated by the presence of five aromatic aminotransferases. Each aminotransferase is capable of transamination in vitro with any of the three keto acid intermediates in the aromatic pathway (phenylpyruvate, 4-hydroxyphenylpyruvate, or prephenate). The fractional contribution of these aminotransferases to particular transamination reactions in vivo can best be approached through the systematic and sequential elimination of individual aminotransferase activities by mutation. A program of sequential mutagenesis has produced two aminotransferase-deficient mutations. The first mutation imposed a phenotype of bradytrophy for L-phenylalanine (doubling time of 2.4 h in minimal salts/glucose medium compared to a 1.0-h doubling time for wild type). This mutant completely lacked an enzyme denoted aminotransferase AT-2. A genetic background of aminotransferase AT-2 deficiency was used to select for a second mutation which produced a phenotype of multiple auxotrophy for L-phenylalanine, L-aspartate, and L-glutamate. The double mutant completely lacked activity for aromatic aminotransferase AT-1 in addition to the missing aminotransferase AT-2. Enzymes AT-1 (Mr = 64,000) and AT-2 (Mr = 50,000) were readily separated from one another by gel filtration and were individually characterized for pH optima, freeze-thaw stability, heat lability, and molecular weight. The phenotypic and enzymological characterizations of the aminotransferase mutants strongly support the primary in vivo role of enzyme AT-2 in L-phenylalanine and L-tyrosine biosynthesis, while enzyme AT-1 must primarily be engaged in L-aspartate and L-glutamate synthesis. The substrate specificities and possible in vivo functions for AT-3, AT-4, and AT-5 are also considered.

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.

Comparative allostery of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase as an indicator of taxonomic relatedness in pseudomonad genera.

Recently, an analysis of the enzymological patterning of L-tyrosine biosynthesis was shown to distinguish five taxonomic groupings among species currently named Pseudomonas, Xanthomonas, or Alcaligenes (Byng et al., J. Bacteriol. 144:247--257, 1980). These groupings paralleled with striking consistency those previously defined by ribosomal ribonucleic acid-deoxyribonucleic acid homology relationships. The comparative allostery of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase has previously been shown to be a useful indicator of taxonomic relationship at about the level of genus. The comparative allostery of DAHP synthetase was evaluated in relationship to data available from the same pseudomonad species previously studied. Species of Xanthomonas and some named species of Pseudomonas, e.g., P. maltophilia, were unmistakably recognized as belonging to group V, having a DAHP synthetase sensitive to sequential feedback inhibition by chorismate. This control pattern is thus far unique to group V pseudomonads among microorganisms. Group V organisms were also unique in their possession of DAHP synthetase enzymes that were unstimulated by divalent cations. Group IV pseudomonads (P. diminuta) were readily distinguished by the retro-tryptophan pattern of control for DAHP synthetase. Activity for DAHP synthetase was not always recovered in group IV species, e.g., P. vesicularis. The remaining three groups exhibited overlapping patterns of DAHP synthetase sensitivity to both L-phenylalanine and L-tyrosine. Individual species cannot be reliably keyed to group I. II, or III without other data. However, each group overall exhibited a different trend of relative sensitivity to L-tyrosine and L-phenylalanine. Thus, although enzymological patterning of L-tyrosine biosynthesis alone can be used to separate the five pseudomonad groups, the independent assay of DAHP synthetase control pattern can be used to confirm assignments. The latter approach is, in fact, the easiest and most definitive method for recognition of group V (and often of group IV) species.

Variable enzymological patterning in tyrosine biosynthesis as a means of determining natural relatedness among the Pseudomonadaceae.

Enzymes of tyrosine biosynthesis (prephenate dehydrogenase and arogenate dehydrogenase) were characterized in 90 species currently classified within the genera Pseudomonas, Xanthomonas, and Alcaligenes. Variation in cofactor specificity and regulatory properties of the dehydrogenase proteins allowed the separation of five groups. Taxa defined by enzymological patterning corresponded strikingly with the five ribosomal ribonucleic acid (rRNA) homology groups established via rRNA-deoxyribonucleic acid hybridization. rRNA homology groups I, IV, and V all lack activity for arogenate/nicotinamide adenine dinucleotide phosphate (NADP) dehydrogenase and separated on this criterion from groups II and III, which have the activity. Group II species possess arogenate dehydrogenase enzyme (reactive with either NAD or NADP) sensitive to feedback inhibition by tyrosine, thereby separating from group III species whose corresponding enzyme was totally insensitive to feedback inhibition. The presence of prephenate/NADP dehydrogenase in group IV defined its separation from groups I and V, which lack this enzyme activity. Group I species possess an arogenate/NAD dehydrogenase that was highly sensitive to inhibition by tyrosine and a prephenate/NAD dehydrogenase of relative insensitivity to tyrosine inhibition. The opposite pattern of sensitivity/insensitivity was seen in group V species. These dehydrogenase characterizations are highly reliable for the keying of a given species to one of the five rRNA homology groups. If necessary, other confirmatory assays can be included using other aromatic pathway enzymes. These results further document the validity and utility of the approach of comparative enzymology and allostery for classification of microorganisms.

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.

Diverse enzymological patterns of phenylalanine biosynthesis in pseudomonads are conserved in parallel with deoxyribonucleic acid homology groupings.

l-Tyrosine biosynthesis in nature has proven to be an exceedingly diverse gestalt of variable biochemical routing, cofactor specificity of pathway dehydrogenases, and regulation. A detailed analysis of this enzymological patterning of l-tyrosine biosynthesis formed a basis for the clean separation of five taxa among species currently named Pseudomonas, Xanthomonas, or Alcaligenes (Byng et al., J. Bacteriol. 144:247-257, 1980). These groupings paralleled taxa established independently by ribosomal ribonucleic acid/deoxyribonucleic acid (DNA) homology relationships. It was later found that the distinctive allosteric control of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in group V, a group dominated by most named species of Xanthomonas (Whitaker et al., J. Bacteriol. 145:752-759, 1981), was the most striking and convenient criterion of group V identity. Diversity in the biochemical routing of l-phenylalanine biosynthesis and regulation was also found, and phenylalanine patterning is in fact the best single enzymatic indicator of group IV (Pseudomonas diminuta and Pseudomonas vesicularis) identity. Enzymological patterning of l-phenylalanine biosynthesis allowed discrimination of still finer groupings consistently paralleling that achieved by the criterion of DNA/DNA hybridization. Accordingly, the five ribosomal ribonucleic acid/DNA homology groups further separate into eight DNA homology subgroups and into nine subgroups based upon phenylalanine pathway enzyme profiling. (Although both fluorescent and nonfluorescent species of group I pseudomonads fall into a common DNA homology group, fluorescent species were distinct from nonfluorescent species in our analysis.) Hence, phenylalanine patterning data provide a relatively fine-tuned probe of hierarchical level. The combined application of these various enzymological characterizations, feasibly carried out in crude extracts, offers a comprehensive and reliable definition of 11 pseudomonad subgroups, 2 of them being represented by species of Alcaligenes.

The evolutionary pattern of aromatic amino acid biosynthesis and the emerging phylogeny of pseudomonad bacteria.

Pseudomonad bacteria are a phylogenetically diverse assemblage of species named within contemporary genera that include Pseudomonas, Xanthomonas and Alcaligenes. Thus far, five distinct rRNA homology groups (Groups I through V) have been established by oligonucleotide cataloging and by rRNA/DNA hybridization. A pattern of enzymic features of aromatic amino acid biosynthesis (enzymological patterning) is conserved at the level of rRNA homology, five distinct and unambiguous patterns therefore existing in correspondence with the rRNA homology groups. We sorted 87 pseudomonad strains into Groups (and Subgroups) by aromatic pathway patterning. The reliability of this methodology was tested in a blind study using coded cultures of diverse pseudomonad organisms provided by American type Culture Collection. Fourteen of 14 correct assignments were made at the Group level (the level of rRNA homology), and 12 of 14 correct assignments were made at the finer-tuned Subgroup levels. Many strains of unknown rRNA-homology affiliation had been placed into tentative rRNA groupings based upon enzymological patterning. Positive confirmation of such strains as members of the predicted rRNA homology groups was demonstrated by DNA/rRNA hybridization in nearly every case. It seems clear that the combination of these molecular approaches will make it feasible to deduce the evolution of biochemical-pathway construction and regulation in parallel with the emerging phylogenies of microbes housing these pathways.