Biochemical and genetic characterization of an early step in a novel pathway for the biosynthesis of aromatic amino acids and p-aminobenzoic acid in the archaeon Methanococcus maripaludis

Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon and facultative autotroph capable of biosynthesizing all the amino acids and vitamins required for growth. In this work, the novel 6-deoxy-5-ketofructose-1-phosphate (DKFP) pathway for the biosynthesis of aromatic amino acids (AroAAs) and p-aminobenzoic acid (PABA) was demonstrated in M. maripaludis. Moreover, PABA was shown to be derived from an early intermediate in AroAA biosynthesis and not from chorismate. Following metabolic labelling with [U-(13)C]-acetate, the expected enrichments for phenylalanine and arylamine derived from PABA were observed. DKFP pathway activity was reduced following growth with aryl acids, an alternative source of the AroAAs. Lastly, a deletion mutant of aroA', which encodes the first step in the DKFP pathway, required AroAAs and PABA for growth. Complementation of the mutants by an aroA' expression vector restored the wild-type phenotype. In contrast, a deletion of aroB', which encodes the second step in the DKFP pathway, did not require AroAAs or PABA for growth. Presumably, methanococci contain an alternative activity for this step. These results identify the initial reactions of a new pathway for the biosynthesis of PABA in methanococci.     

Genetic analysis of the upper phenylacetate catabolic pathway in the production of tropodithietic acid by Phaeobacter gallaeciensis

Production of the antibiotic tropodithietic acid (TDA) depends on the central phenylacetate catabolic pathway, specifically on the oxygenase PaaABCDE, which catalyzes epoxidation of phenylacetyl-coenzyme A (CoA). Our study was focused on genes of the upper part of this pathway leading to phenylacetyl-CoA as precursor for TDA. Phaeobacter gallaeciensis DSM 17395 encodes two genes with homology to phenylacetyl-CoA ligases (paaK1 and paaK2), which were shown to be essential for phenylacetate catabolism but not for TDA biosynthesis and phenylalanine degradation. Thus, in P. gallaeciensis another enzyme must produce phenylacetyl-CoA from phenylalanine. Using random transposon insertion mutagenesis of a paaK1-paaK2 double mutant we identified a gene (ior1) with similarity to iorA and iorB in archaea, encoding an indolepyruvate:ferredoxin oxidoreductase (IOR). The ior1 mutant was unable to grow on phenylalanine, and production of TDA was significantly reduced compared to the wild-type level (60%). Nuclear magnetic resonance (NMR) spectroscopic investigations using (13)C-labeled phenylalanine isotopomers demonstrated that phenylalanine is transformed into phenylacetyl-CoA by Ior1. Using quantitative real-time PCR, we could show that expression of ior1 depends on the adjacent regulator IorR. Growth on phenylalanine promotes production of TDA, induces expression of ior1 (27-fold) and paaK1 (61-fold), and regulates the production of TDA. Phylogenetic analysis showed that the aerobic type of IOR as found in many roseobacters is common within a number of different phylogenetic groups of aerobic bacteria such as Burkholderia, Cupriavidis, and Rhizobia, where it may also contribute to the degradation of phenylalanine.     

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.     

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.     

Control of aromatic acid biosynthesis in Bacillus subtilis: sequenial feedback inhibition

Nester, E. W. (University of Washington, Seattle), and R. A. Jensen. Control of aromatic acid biosynthesis in Bacillus subtilis: sequential feedback inhibition. J. Bacteriol. 91:1594-1598. 1966.-The three major end products of aromatic acid synthesis, tyrosine, phenylalanine, and tryptophan, were tested for their ability to inhibit the first enzymes of the three terminal branches of the pathway as well as the enzyme common to both tyrosine and phenylalanine synthesis. Tyrosine inhibits the activity of prephenate dehydrogenase and also prephenate dehydratase to a limited extent. Phenylalanine inhibits the activity of prephenate dehydratase and, at much higher concentrations, prephenate dehydrogenase. Tryptophan inhibits the activity of anthranilate synthetase and, to some extent, prephenate dehydrogenase and prephenate dehydratase. Chorismate mutase is not inhibited by either 1 mm tyrosine or 1 mm phenylalanine when these are present singly or together in the reaction mixture. The significance of the feedback control of the terminal branches to the feedback control of that part of the pathway common to the synthesis of all three amino acids is discussed.     

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.     

Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16. II. The isolation and characterization of mutants auxotrophic for phenylalanine and tyrosine.

1. Mutants derived from the hydrogen bacterium Alcaligenes eutrophus strain H 16 auxotrophic for phenylalanine and tyrosine were isolated employing mutagenic agents (EMS, nitrite), the colistine counterselection technique and the "pin-point" isolation method. Three different types of mutants were found: (1) Mutants, requiring phenylalanine or phenylpyruvate for growth, were affected in chorismate mutase as well as prephenate dehydratase. Both activities were regained by reversion to prototrophy. The auxotrophic strains accumulated chorismic acid. (2) Strains with a growth response similar to that of the first group lacked only prephenate dehydratase activity which was partially regained by reversion. Chorismate mutase and prephenate dehydrogenase were derepressed up to two-fold. Mutants grown in minimal medium excreted prephenic acid. (3) The third type of mutants required phenylalanine or phenylpyruvate and grew slowly when supplemented with chorismate or prephenate. The enzymes involved in the specific pathway of phenylalanine and tyrosine were found to be present. Some of them were even more active than in the wild-type. 2. Mutants accumulating chorismic acid or prepheric acid were able to grow on minimal medium when incubated long enough. The chemical instability of the excretion products resulted in their nonenzymatic conversion to subsequent intermediates which were taken up by the cells, allowing growth. 3. A method is described for preparing barium prephenate using the auxotrophic mutant 6B-1 derived from A.eutrophus H 16. Prephenic acid, excreted by this strain, was obtained from the culture filtrate with a purity of at least 70% and a yield of approximately 180 mg per 21 of medium.     

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.     

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.     

Involvement of quinolinate phosphoribosyl transferase in promotion of potato growth by a Burkholderia strain

Burkholderia sp. strain PsJN stimulates root growth of potato explants compared to uninoculated controls under gnotobiotic conditions. In order to determine the mechanism by which this growth stimulation occurs, we used Tn5 mutagenesis to produce a mutant, H41, which exhibited no growth-promoting activity but was able to colonize potato plants as well as the wild-type strain. The gene associated with the loss of growth promotion in H41 was shown to exhibit 65% identity at the amino acid level to the nadC gene encoding quinolinate phosphoribosyltransferase (QAPRTase) in Ralstonia solanacearum. Complementation of H41 with QAPRTase restored growth promotion of potato explants by this mutant. Expression of the gene identified in Escherichia coli yielded a protein with QAPRTase activities that catalyzed the de novo formation of nicotinic acid mononucleotide (NaMN). Two other genes involved in the same enzymatic pathway, nadA and nadB, were physically linked to nadC. The nadA gene was cotranscribed with nadC as an operon in wild-type strain PsJN, while the nadB gene was located downstream of the nadA-nadC operon. Growth promotion by H41 was fully restored by addition of NaMN to the tissue culture medium. These data suggested that QAPRTase may play a role in the signal pathway for promotion of plant growth by PsJN.     

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.     

Biosynthesis of aromatic amino acids in Nocardia sp. 239: effects of amino acid analogues on growth and regulatory enzymes

Summary Further steps required for overproduction of aromatic amino acids by a mutant strain of Nocardia sp. 239 (Noc 87-13), unable to grow on l-phenylalanine as a sole carbon and energy source, were investigated. A number of analogues of the aromatic amino acids displayed severe inhibitory effects on the activities of regulatory enzymes in the biosynthetic pathway and growth of the organism in glucose mineral medium. l-Tryptophane analogues strongly inhibited 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity. l-Tyrosine analogues especially inhibited DAHP synthase and chorismate mutase, whereas l-phenylalanine analogues strongly inhibited chorismate mutase and prephenate dehydratase activity. Addition of the aromatic amino acids and their precursors chorismate, 4-hydroxyphenylpyruvate, phenylpyruvate and anthranilate, to the medium counteracted the growth inhibitory effect of specific analogues. The data indicate that ortho- (OFP) and para-fluoro-d,l-phenylalanine (PFP), and l-phenylalanine amide, are the most suitable analogues for the isolation of feedback-inhibition-insensitive prephenate dehydratase mutants. Attempts to isolate l-tyrosine and l-trytophane auxotrophic mutants were only successful in the latter case, resulting in the selection of a stable anthranilate synthase-negative mutant (Noc 87-13-14). Uptake of aromatic amino acids in Nocardia sp. 239 most likely involves a common transport system. This necessitates the use of anthranilate, rather than l-trytophane, as a supplement during the isolation of l-tyrosine auxotrophic and OFP- and/or PFP-resistant mutant derivative strains of Noc 87-13-14. Offprint requests to: L. Dijkhuizen     

An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria

The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase. Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns. A new extremehalophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria. In addition to the conventional sensitivity to feedback inhibition by l-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors. Thus, l-tryptophan inhibited activity while l-tyrosine, l-methionine, l-leucine, and l-isoleucine activated the enzyme. l-Isoleucine and l-phenylalanine were effective at M levels; other effectors operated at mM levels. A regulatory mutant selected for resistance to growth inhibition caused by -2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished. Inhibition by l-tryptophan was also lost, and activation by allosteric activators was diminished. Not only was sensitivity to feedback inhibition by l-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis). It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster.     

Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>

Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine (mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 M phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in K _m and k _cat values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the K _m value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the K _m and k _cat values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity.     

Identification of a novel mycobacterial 3-hydroxyacyl-thioester dehydratase, HtdZ (Rv0130), by functional complementation in yeast

We report on the identification of Mycobacterium tuberculosis HtdZ (Rv0130), representing a novel 3-hydroxyacyl-thioester dehydratase. HtdZ was picked up by the functional complementation of Saccharomyces cerevisiae htd2Delta cells lacking the dehydratase of mitochondrial type II fatty acid synthase. Mutant cells expressing HtdZ contained dehydratase activity, recovered their respiratory ability, and partially restored de novo lipoic acid synthesis.     

Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16

1. Mutants derived from the hydrogen bacterium Alcaligenes eutrophus strain H16 auxotrophic for phenylalanine and tyrosine were isolated employing mutagenic agents (EMS, nitrite), the colistine counterselection technique and the “pin-point” isolation method. Three different types of mutants were found: (1) Mutants, requiring phenylalanine or phenylpyruvate for growth, were affected in chorismate mutase as well as prephenate dehydratase. Both activities were regained by reversion to prototrophy. The auxotrophic strains accumulated chorismic acid. (2) Strains with a growth response similar to that of the first group lacked only prephenate dehydratase activity which was partially regained by reversion. Chorismate mutase and prephenate dehydrogenase were derepressed up to two-fold. Mutants grown in minimal medium excreted prephenic acid. (3) The third type of mutants required phenylalanine or phenylpyruvate and grew slowly when supplemented with chorismate or prephenate. The enzymes involved in the specific pathway of phenylalanine and tyrosine were found to be present. Some of them were even more active than in the wild-type.
2. Mutants accumulating chorismic acid or prephenic acid were able to grow on minimal medium when incubated long enough. The chemical instability of the excretion products resulted in their nonenzymatic conversion to subsequent intermediates which were taken up by the cells, allowing growth.
3. A method is described for preparing barium prephenate using the auxotrophic mutant 6B-1 derived from A. eutrophus H16. Prephenic acid, excreted by this strain, was obtained from the culture filtrate with a purity of at least 70% and a yield of approximately 180 mg per 2 l of medium.

     

Production of l-Phenylalanine from Starch by Analog-Resistant Mutants of Bacillus polymyxa

p-Fluorophenylalanine-resistant mutants of starch-degrading Bacillus polymyxa ATCC 842, generated by ethyl methanesulfonate mutagenesis followed by incubation with caffeine, overproduced small amounts of l-phenylalanine (l-phe) from starch. A beta-2-thienylalanine-resistant mutant (BT-7) derived from p-fluorophenylalanine mutant (C-4000 FP-4) and resistant to both p-fluorophenylalanine and beta-2-thienylalanine produced 0.5 g of l-phe and 0.15 g of l-tyrosine per liter from 10 g of starch per liter when growing in a minimal medium. trans-Cinnamic acid (CA) was also excreted by both mutants, indicating the possibility of l-phenylalanine ammonia-lyase-induced deamination of l-phe to CA. The amount of l-phe-derived CA detected in BT-7 was less compared with mutant C-4000 FP-4. CA production was induced in the parent only when l-phe was used as a sole nitrogen source. Time of CA production in the two mutants could be delayed by addition of other nitrogen sources, an indication of possible l-phenylalanine ammonia-lyase inhibition or repression. The presence of l-phenylalanine ammonia-lyase in B. polymyxa mutant C-4000 FP-4 was confirmed by assays of cell-free extracts from cells grown in starch minimal medium containing l-phe as the sole nitrogen source. Preliminary studies of the regulation of deoxy-d-arabino-heptulosonate-7-phosphate synthase and prephenate dehydratase in the wild-type strain showed that deoxy-d-arabino-heptulosonate-7-phosphate synthase was subject to feedback inhibition by l-phe, l-tyrosine, and l-tryptophan. Inhibition by each amino acid was to a similar extent singly or in combination at a 0.5 mM level of each amino acid. Prephenate dehydratase was feedback inhibited by l-phe, but not by l-tyrosine or l-tryptophan or both. In the double analog-resistant mutant BT-7, deoxy-d-arabino-heptulosonate-7-phosphate synthase had specific activity similar to that in the wild type, and the enzyme was still subject to feedback inhibition. However, prephenate dehydratase had increased specific activity and it was also insensitive to feedback inhibition by l-phe. The overproduction of aromatic amino acids by BT-7 was thought to be due, at least in part, to deregulation of feedback inhibition of prephenate dehydratase. Chorismate mutase was not subject to feedback inhibition in the wild type and was unaffected in the mutant.     

Regulation of phenylalanine biosynthesis in Rhodotorula glutinis.

The phenylalanine biosynthetic pathway in the yeast Rhodotorula glutinis was examined, and the following results were obtained. (i) 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase in crude extracts was partially inhibited by tyrosine, tryptophan, or phenylalanine. In the presence of all three aromatic amino acids an additive pattern of enzyme inhibition was observed, suggesting the existence of three differentially regulated species of DAHP synthase. Two distinctly regulated isozymes inhibited by tyrosine or tryptophan and designated DAHP synthase-Tyr and DAHP synthase-Trp, respectively, were resolved by DEAE-Sephacel chromatography, along with a third labile activity inhibited by phenylalanine tentatively identified as DAHP synthase-Phe. The tyrosine and tryptophan isozymes were relatively stable and were inhibited 80 and 90% by 50 microM of the respective amino acids. DAHP synthase-Phe, however, proved to be an extremely labile activity, thereby preventing any detailed regulatory studies on the partially purified enzyme. (ii) Two species of chorismate mutase, designated CMI and CMII, were resolved in the same chromatographic step. The activity of CMI was inhibited by tyrosine and stimulated by tryptophan, whereas CMII appeared to be unregulated. (iii) Single species of prephenate dehydratase and phenylpyruvate aminotransferase were observed. Interestingly, the branch-point enzyme prephenate dehydratase was not inhibited by phenylalanine or affected by tyrosine, tryptophan, or both. (iv) The only site for control of phenylalanine biosynthesis appeared to be DAHP synthase-Phe. This is apparently sufficient since a spontaneous mutant, designated FP9, resistant to the growth-inhibitory phenylalanine analog p-fluorophenylalanine contained a feedback-resistant DAHP synthase-Phe and cross-fed a phenylalanine auxotroph of Bacillus subtilis.     

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.     

Mutation of a rice gene encoding a phenylalanine biosynthetic enzyme results in accumulation of phenylalanine and tryptophan

Two distinct biosynthetic pathways for Phe in plants have been proposed: conversion of prephenate to Phe via phenylpyruvate or arogenate. The reactions catalyzed by prephenate dehydratase (PDT) and arogenate dehydratase (ADT) contribute to these respective pathways. The Mtr1 mutant of rice (Oryza sativa) manifests accumulation of Phe, Trp, and several phenylpropanoids, suggesting a link between the synthesis of Phe and Trp. Here, we show that the Mtr1 mutant gene (mtr1-D) encodes a form of rice PDT with a point mutation in the putative allosteric regulatory region of the protein. Transformed callus lines expressing mtr1-D exhibited all the characteristics of Mtr1 callus tissue. Biochemical analysis revealed that rice PDT possesses both PDT and ADT activities, with a preference for arogenate as substrate, suggesting that it functions primarily as an ADT. The wild-type enzyme is feedback regulated by Phe, whereas the mutant enzyme showed a reduced feedback sensitivity, resulting in Phe accumulation. In addition, these observations indicate that rice PDT is critical for regulating the size of the Phe pool in plant cells. Feeding external Phe to wild-type callus tissue and seedlings resulted in Trp accumulation, demonstrating a connection between Phe accumulation and Trp pool size.