Interconvertible molecular-weight forms of the bifunctional chorismate mutase-prephenate dehydratase from Acinetobacter calcoaceticus.

Acinetobacter calcoaceticus belongs to a large phylogenetic cluster of gram-negative procaryotes that all utilize a bifunctional P-protein (chorismate mutase-prephenate dehydratase) [EC 5.4.99.5-4.2.1.51] for phenylalanine biosynthesis. These two enzyme activities from Ac. calcoaceticus were inseparable by gel-filtration or DEAE-cellulose chromatography. The molecular weight of the P-protein in the absence of effectors was 65,000. In the presence of L-tyrosine (dehydratase activator) or L-phenylalanine (inhibitor of both P-protein activities), the molecular weight increased to 122,000. Maximal activation (23-fold) of prephenate dehydratase was achieved at 0.85 mM L-tyrosine. Under these conditions, dehydratase activity exhibited a hysteretic response to increasing protein concentration. Substrate saturation curves for prephenate dehydratase were hyperbolic at L-tyrosine concentrations sufficient to give maximal activation (yielding a Km,app of 0.52 mM for prephenate), whereas at lower L-tyrosine concentrations the curves were sigmoidal. Dehydratase activity was inhibited by L-phenylalanine, and exhibited cooperative interactions for inhibitor binding. A Hill plot yielded an n' value of 3.1. Double-reciprocal plots of substrate saturation data obtained in the presence of L-phenylalanine indicated cooperative interactions for prephenate in the presence of inhibitor. The n values obtained were 1.4 and 3.0 in the absence or presence of 0.3 mM L-phenylalanine, respectively. The hysteretic response of chorismate mutase activity to increasing enzyme concentration was less dramatic than that of prephenate dehydratase. A Km,app for chorismate of 0.63 mM was obtained. L-Tyrosine did not affect chorismate mutase activity, but mutase activity was inhibited both by L-phenylalanine and by prephenate. Interpretations are given about the physiological significance of the overall pattern of allosteric control of the P-protein, and the relationship between this control and the effector-induced molecular-weight transitions. The properties of the P-protein in Acinetobacter are considered within the context of the ubiquity of the P-protein within the phylogenetic cluster to which this genus belongs.

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 important 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 (greater than 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 and L-arogenate.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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)

A pair of regulatory isozymes for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase is conserved within group I pseudomonads.

Two closely related subgroups of group I pseudomonads, which differ from one another in the overall enzymatic makeup of aromatic amino acid biosynthesis, possess in common the recently characterized major (tyrosine-sensitive) and minor (tryptophan-sensitive) isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Pseudomonas aeruginosa (17). Since these characterizations were made for strains whose phylogenetic positions have been determined by oligonucleotide cataloging, an initial perception of the evolution of aromatic pathway construction and regulation is emerging.

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.

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.

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 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.

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.

Biosynthesis of phenazine pigments in mutant and wild-type cultures of Pseudomonas aeruginosa.

Pigmentation mutants of Pseudomonas aeruginosa, selected by observed visual differences in coloration from the wild-type strain, were examined for altered patterns of phenazine synthesis. Three classes of mutants that were incapable of pyocyanine production were identified. Pigmentation patterns that were found to characterize the various mutant classes implicated precursor-product relationships, and a biochemical scheme covering the terminal reactions of pyocyanine biosynthesis is proposed. Among compounds tested as inhibitors of pigmentation, two effectively inhibited pyocyanine production production while allowing cell growth. p-Aminobenzoate inhibited total pigmentation; i.e., no other phenazine accumulated. m-Aminobenzoate inhibited a presumptive methylation step in pyocyanine biosynthesis, abolishing the formation of pyocyanine and aeruginosin pigments but increasing the yields of phenazine 1-carboxylic acid and oxychlororaphin. D-[2,3,4,5(n)-14C]shikimate was most efficiently incorporated into phenazines in the middle to late exponential phase of growth. Label was incorporated predominantly into pyocyanine in the absence of inhibitors and into phenazine 1-carboxylic acid when the organism was grown in the presence of m-aminobenzoate.

Incorporation of [14C]shikimate into plenazines and their further metabolism by Pseudomonas phenazinium.

1. During growth of Pseudomonas phenazinium on l-threonine medium, phenazine pigment formation commenced early and 1,6-dihydroxyphenazine 5,10-dioxide (iodinin) was the major component. Growth on l-[U-(14)C]threonine showed that when growth was complete about 25% of the label had been incorporated into phenazines and 30% into cell substance. 2. The addition of d-[2,3,4,5(n)-(14)C]shikimate to cultures at different phases of growth showed that the greatest efficiency of incorporation (about 70%) occurred in the mid- to late-exponential phase. Phenazines accounting for most of the (14)C supplied were iodinin and 9-hydroxyphenazine-1-carboxylate plus 2,9-dihydroxyphenazine-1-carboxylate. Radioactivity incorporated into cell substance was about one-third of the amount found in phenazines. 3. Kinetic studies showed that radioactivity from a pulse of [(14)C]-shikimate was incorporated into phenazines immediately, without a discernible lag, and into all detectable phenazines simultaneously rather than sequentially. 4. Radioactive phenazines isolated from culture media were fed to growing cultures and their metabolism was studied. The results supported a scheme for the biosynthesis of iodinin and 1,8-dihydroxyphenazine 10-monoxide by a branched pathway. 5. It is proposed that phenazine-1,6-dicarboxylate is the common precursor of all naturally occurring phenazines.

Isolation of pigmentation mutants of Pseudomonas phenazinium.

Pigmentation mutants of Pseudomonas phenazinium unable to synthesize iodinin, or producing it only in reduced amounts, were isolated. The abilities of the mutants to synthesize nine other phenazines were also altered. Cross-feeding experiments and the altered patterns of pigment production suggested metabolic relationships between the phenazine pigments, and a scheme for their biosynthesis is proposed.