Chorismate Mutase-prephenate Dehydratase Research Articles

Bacterial bifunctional chorismate mutase-prephenate dehydratase PheA increases flux into the yeast phenylalanine pathway and improves mandelic acid production

Mandelic acid is an important aromatic fine chemical and is currently mainly produced via chemical synthesis. Recently, mandelic acid production was achieved by microbial fermentations using engineered Escherichia coli and Saccharomyces cerevisiae expressing heterologous hydroxymandelate synthases (hmaS). The best-performing strains carried a deletion of the gene encoding the first enzyme of the tyrosine biosynthetic pathway and therefore were auxotrophic for tyrosine. This was necessary to avoid formation of the competing intermediate hydroxyphenylpyruvate, the preferred substrate for HmaS, which would have resulted in the predominant production of hydroxymandelic acid. However, feeding tyrosine to the medium would increase fermentation costs. In order to engineer a tyrosine prototrophic mandelic acid-producing S. cerevisiae strain, we tested three strategies: (1) rational engineering of the HmaS active site for reduced binding of hydroxyphenylpyruvate, (2) compartmentalization of the mandelic acid biosynthesis pathway by relocating HmaS together with the two upstream enzymes chorismate mutase Aro7 and prephenate dehydratase Pha2 into mitochondria or peroxisomes, and (3) utilizing a feedback-resistant version of the bifunctional E. coli enzyme PheA (PheAfbr) in an aro7 deletion strain. PheA has both chorismate mutase and prephenate dehydratase activity. Whereas the enzyme engineering approaches were only successful in respect to reducing the preference of HmaS for hydroxyphenylpyruvate but not in increasing mandelic acid titers, we could show that strategies (2) and (3) significantly reduced hydroxymandelic acid production in favor of increased mandelic acid production, without causing tyrosine auxotrophy. Using the bifunctional enzyme PheAfbr turned out to be the most promising strategy, and mandelic acid production could be increased 12-fold, yielding titers up to 120 mg/L. Moreover, our results indicate that utilizing PheAfbr also shows promise for other industrial applications with S. cerevisiae that depend on a strong flux into the phenylalanine biosynthetic pathway.

Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy

Flavonoids are valuable natural products widely used in human health and nutrition. Recent advances in synthetic biology and metabolic engineering have yielded improved strain titers and yields. However, current fermentation strategies often require supplementation of expensive phenylpropanoic precursors in the media and separate evaluation of each strategy in turn as part of the flavonoid pathway, implicitly assuming the modifications are additive. In this study, an Escherichia coli fermentation system was developed to bypass both of these problems. An eight-step pathway, consisting of 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydratase (CM/PDT), phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was assembled on four vectors in order to produce the flavonoid precursor (2S)-pinocembrin directly from glucose. Furthermore, a modular metabolic strategy was employed to identify conditions that optimally balance the four pathway modules. Once this metabolic balance was achieved, such strains were capable of producing 40.02mg/L (2S)-pinocembrin directly from glucose. These results were attained by culturing engineered cells in minimal medium without additional precursor supplementation. The fermentation platform described here paves the way for the development of an economical process for microbial production of flavonoids directly from glucose.

Metabolic engineering of Escherichia coli for improving l-3,4-dihydroxyphenylalanine (l-DOPA) synthesis from glucose

L-3,4-dihydroxyphenylalanine (L-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of L-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of L-tyrosine (L-Tyr), an L-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on L-Tyr production of PTS inactivation (PTS(-) gluc(+) phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of L-Tyr production (q( L-Tyr)), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q( L-Tyr) in the PTS(+) and the PTS(-) gluc(+) strains, respectively. An 8.6-fold increase in L-Tyr yield from glucose was observed in the PTS(-) gluc(+) tyrR (-) strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for L-Tyr production caused the synthesis of L-DOPA. One of such strains, having the PTS(-) gluc(+) tyrR (-) phenotype, displayed the best production parameters in minimal medium, with a specific rate of L-DOPA production of 13.6 mg/g/h, L-DOPA yield from glucose of 51.7 mg/g and a final L-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of L-DOPA in 50 h.

Enhanced l-phenylalanine biosynthesis by co-expression of pheA fbr and aroF wt

A β-2-thienylalanine-resistant E. coli K12 mutant carrying a Thr326Pro mutation in the regulation (R) domain of pheA ( pheA fbr ) was obtained by N-methyl-N′-nitro-N-nitrosoguanidine (NTG) mutagenesis. In the presence of 200 mM l-phenylalanine ( l-Phe), a recombinant E. coli WSH-Z06 (pAP-B03) carrying pheA fbr as well as wild-type aroF ( aroF wt ) exhibited more than 70% of the chorismate mutase-prephenate dehydratase (CM–PDT) activity as observed in the absence of this amino acid. The l-Phe titer of WSH-Z06 (pAP-B03) reached 35.38 g/L in a 3-L fermentor, which was 2.81-fold higher than that of the original strain E. coli WSH-Z06. Furthermore, the l-Phe yield on glucose of WSH-Z06 (pAP-B03) (0.26 mol/mol) was twice that of E. coli WSH-Z06. This recombinant E. coli WSH-Z06 (pAP-B03) is a potential strain for over-production of l-Phe.

Metabolic Engineering of Escherichia coli for l -Tyrosine Production by Expression of Genes Coding for the Chorismate Mutase Domain of the Native Chorismate Mutase-Prephenate Dehydratase and a Cyclohexadienyl Dehydrogenase from Zymomonas mobilis

The expression of the feedback inhibition-insensitive enzyme cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase domain from native chorismate mutase-prephenate dehydratase (PheA(CM)) from Escherichia coli was compared to the expression of native feedback inhibition-sensitive chorismate mutase-prephenate dehydrogenase (CM-TyrA(p)) with regard to the capacity to produce l-tyrosine in E. coli strains modified to increase the carbon flow to chorismate. Shake flask experiments showed that TyrC increased the yield of l-tyrosine from glucose (Y(l-Tyr/Glc)) by 6.8-fold compared to the yield obtained with CM-TyrA(p). In bioreactor experiments, a strain expressing both TyrC and PheA(CM) produced 3 g/liter of l-tyrosine with a Y(l-Tyr/Glc) of 66 mg/g. These values are 46 and 48% higher than the values for a strain expressing only TyrC. The results show that the feedback inhibition-insensitive enzymes can be employed for strain development as part of a metabolic engineering strategy for l-tyrosine production.

Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine

BackgroundThe rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA) of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed.ResultsMTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt) in the best PTS- L-Phe overproducing strain (PB12-ev2). Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to both PTS inactivation and the presence of plasmids. For instance, acs-ackA, sucABCD, and sdhABCD operons were up-regulated in PB12 (PTS mutant that carries an arcB- mutation). The induction of these operons was further increased by the presence of plasmids in PB12-ev2. Some genes involved in the shikimate and specific aromatic amino acid pathways showed down-regulation in the L-Phe overproducing strains, might cause possible metabolic limitations in the shikimate pathway.ConclusionThe identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains.

Structure of Phenylalanine Hydroxylase from Colwellia psychrerythraea 34H, a Monomeric Cold Active Enzyme with Local Flexibility around the Active Site and High Overall Stability

The characteristic of cold-adapted enzymes, high catalytic efficiency at low temperatures, is often associated with low thermostability and high flexibility. In this context, we analyzed the catalytic properties and solved the crystal structure of phenylalanine hydroxylase from the psychrophilic bacterium Colwellia psychrerythraea 34H (CpPAH). CpPAH displays highest activity with tetrahydrobiopterin (BH(4)) as cofactor and at 25 degrees C (15 degrees C above the optimal growth temperature). Although the enzyme is monomeric with a single L-Phe-binding site, the substrate binds cooperatively. In comparison with PAH from mesophilic bacteria and mammalian organisms, CpPAH shows elevated [S(0.5)](L-Phe) (= 1.1 +/- 0.1 mm) and K(m)(BH(4))(= 0.3 +/- 0.1 mm), as well as high catalytic efficiency at 10 degrees C. However, the half-inactivation and denaturation temperature is only slightly lowered (T(m) approximately 52 degrees C; where T(m) is half-denaturation temperature), in contrast to other cold-adapted enzymes. The crystal structure shows regions of local flexibility close to the highly solvent accessible binding sites for BH(4) (Gly(87)/Phe(88)/Gly(89)) and l-Phe (Tyr(114)-Pro(118)). Normal mode and COREX analysis also detect these and other areas with high flexibility. Greater mobility around the active site and disrupted hydrogen bonding abilities for the cofactor appear to represent cold-adaptive properties that do not markedly affect the thermostability of CpPAH.

Exhaustive Mutagenesis of Six Secondary Active-Site Residues in Escherichia coli Chorismate Mutase Shows the Importance of Hydrophobic Side Chains and a Helix N-Capping Position for Stability and Catalysis

Secondary active-site residues in enzymes, including hydrophobic amino acids, may contribute to catalysis through critical interactions that position the reacting molecule, organize hydrogen-bonding residues, and define the electrostatic environment of the active site. To ascertain the tolerance of an important model enzyme to mutation of active-site residues that do not directly hydrogen bond with the reacting molecule, all 19 possible amino acid substitutions were investigated in six positions of the engineered chorismate mutase domain of the Escherichia coli chorismate mutase-prephenate dehydratase. The six secondary active-site residues were selected to clarify results of a previous test of computational enzyme design procedures. Five of the positions encode hydrophobic side chains in the wild-type enzyme, and one forms a helix N-capping interaction as well as a salt bridge with a catalytically essential residue. Each mutant was evaluated for its ability to complement an auxotrophic chorismate mutase deletion strain. Kinetic parameters and thermal stabilities were measured for variants with in vivo activity. Altogether, we find that the enzyme tolerated 34% of the 114 possible substitutions, with a few mutations leading to increases in the catalytic efficiency of the enzyme. The results show the importance of secondary amino acid residues in determining enzymatic activity, and they point to strengths and weaknesses in current computational enzyme design procedures.

Prephenate dehydratase from the aphid endosymbiont (Buchnera) displays changes in the regulatory domain that suggest its desensitization to inhibition by phenylalanine.

Buchnera aphidicola, the prokaryotic endosymbiont of aphids, complements dietary deficiencies with the synthesis and provision of several essential amino acids. We have cloned and sequenced a region of the genome of B. aphidicola isolated from Acyrthosiphon pisum which includes the two-domain aroQ/pheA gene. This gene encodes the bifunctional chorismate mutase-prephenate dehydratase protein, which plays a central role in L-phenylalanine biosynthesis. Two changes involved in the overproduction of this amino acid have been detected. First, the absence of an attenuator region suggests a constitutive expression of this gene. Second, the regulatory domain of the Buchnera prephenate dehydratase shows changes in the ESRP sequence, which is involved in the allosteric binding of phenylalanine and is strongly conserved in prephenate dehydratase proteins from practically all known organisms. These changes suggest the desensitization of the enzyme to inhibition by phenylalanine and would permit the bacterial endosymbiont to overproduce phenylalanine.

Bacillus subtilis chorismate mutase is partially diffusion‐controlled

The effect of viscosogens on the enzyme-catalyzed rearrangement of chorismate to prephenate has been studied. The steady-state parameters kcat and kcat/Km for the monofunctional chorismate mutase from Bacillus subtilis (BsCM) decreased significantly with increasing concentrations of glycerol, whereas the 'sluggish' BsCM mutants C75A and C75S were insensitive to changes in microviscosity. The latter results rule out extraneous interactions of the viscosogen as an explanation for the effects observed with the wild-type enzyme. Additional control experiments show that neither viscosogen-induced shifts in the pH-dependence of the enzyme-catalyzed reaction nor small perturbations of the conformational equilibrium of chorismate can account for the observed effects. Instead, BsCM appears to be limited by substrate binding and product release at low and high substrate concentrations, respectively. Analysis of the kinetic data indicates that diffusive transition states are between 30 and 40% rate-determining in these concentration regimes; the chemical step must contribute to the remaining kinetic barrier. The relatively low value of the 'on' rates for chorismate and prephenate (approximately 2 x 106 m-1.s-1) probably reflects the need for a rare conformation of the enzyme, the ligand, or both for successful binding. Interestingly, the chorismate mutase domain of the bifunctional chorismate mutase-prephenate dehydratase from Escherichia coli, which has steady-state kinetic parameters comparable to those of BsCM but has a much less accessible active site, is insensitive to changes in viscosity and the reaction it catalyses is not diffusion-controlled.

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 m m 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 K m,app of 0.52 m m 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 m m l-phenyl-alanine, respectively. The hysteretic response of chorismate mutase activity to increasing enzyme concentration was less dramatic than that of prephenate dehydratase. A K m,app for chorismate of 0.63 m m 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)

Enzyme-enzyme interaction and the biosynthesis of aromatic amino acids in Escherichia coli

The technique of affinity chromatography has been used to demonstrate that enzymes involved in the biosynthesis of tyrosine and phenylalanine in Escherichia coli undergo reversible interactions. Thus it has been shown that the aromatic amino acid aminotransferase (aromatic-amino-acid: 2-oxoglutarate amino-transferase, EC 2.6.1.57) reacts specifically with chorismate mutase-prephenate dehydrogenase (chorismate pyruvate mutase, EC 5.4.99.5 and prephenate: NAD + oxidoreductase (decarboxylating), EC 1.3.1.12) in the absence of reactants and with chorismate mutase-prephenatedehydratase (prephenate hydro-lyase (decarboxylating), EC 4.2.1.51) in the presence of phyenylpyruvate. Tyrosine causes dissociation of the aminotransferase: mutase-dehydrogenase complex while dissociation of the aminotransferase-mutase-dehydratase complex occurs on omission of phenylpyruvate. Only the active form of chorismate mutase-prephenate dehydrogenase participates in complex formation.

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

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

Regulation of Chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from alcaligenes eutrophus.

Highly purified enzymes from Alcaligenes eutrophus H 16 were used for kinetic studies. Chorismate mutase was feedback inhibited by phenylalanine. In the absence of the inhibitor, the double-reciprocal plot was linear, yielding a Km for chorismate of 0.2 mM. When phenylalanine was present, a pronounced deviation from the Michaelis-Menten hyperbola occurred. The Hill coefficient (n) was 1.7, and Hill plots of velocity versus inhibitor concentrations resulted in a value of n' = 2.3, indicating positive cooperativity. Chorismate mutase was also inhibited by prephenate, which caused downward double-reciprocal plots and a Hill coefficient of n = 0.7, evidence for negative cooperativity. The pH optimum of chorismate mutase ranged from 7.8 to 8.2; its temperature optimum was 47 C. Prephenate dehydratase was competitively inhibited by phenylalanine and activated by tyrosine. Tyrosine stimulated its activity up to 10-fold and decreased the Km for prephenate, which was 0.67 mM without effectors. Tryptophan inhibited the enzyme competitively. Its inhibition constant (Ki = 23 muM) was almost 10-fold higher than that determined for phenylalanine (Ki = 2.6 muM). The pH optimum of prephenate dehydratase was pH 5.7; the temperature optimum was 48 C. Prephenate dehydrogenase was feedback inhibited by tyrosine. Inhibition was competitive with prephenate (Ki = 0.06 mM) and noncompetitive with nicotinamide adenine dinucleotide. The enzyme was further subject to product inhibition by p-hydroxyphenylpyruvate (Ki = 0.13 mM). Its Km for prephenate was 0.045 mM, and that for nicotinamide adenine dinucleotide was 0.14 mM. The pH optimum ranged between 7.0 and 7.6; the temperature optimum was 38 C. It is shown how the sensitive regulation of the entire enzyme system leads to a well-balanced amino acid production.

Purification and properties of chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from Alcaligenes eutrophus.

Chorismate mutase and prephenate dehydratase from Alcaligenes autophus H16 were purified 470-fold with a yield of 24%. During the course of purification, including chromatography on diethylaminoethyl (DEAE)-cellulose, phenylalanine-substituted Sepharose, Sephadex G-200 and hydrogyapatite, both enzymes appeared in association. The ratio of their specific activities remained almost constant. The molecular weight of chorismate mutase-prephenast dehydratase varied from 144,000 to 187,000 due to the three different determination methods used. Treatment of electrophoretically homogeneous mutase-dehydratase with sodium dodecyl sulfate dissociated the enzyme into a single component of molecular weight 47,000, indicating a tetramer of identical subunits. The isoelectric point of the bifunctional enzyme was 5.8. Prephenate dehydrogenase was not associated with other enzyme activities; it was separated from mutasedehydratase by DEAE-cellulose chromatgraphy. Chromatography on DEAE Sephadex, Sephadex G-200, and hydroxyapatite resulted in a 740-fold purification with a yield of 10%. The molecular weight of the enzyme was 55,000 as determined by sucrose gradient centrifugation and 65,000 as determined by gel filtration or electrophoresis. Its isoelectric point was pH 6.6. In the overall conversion of chorismate to phenylpyruvate, free prephenate was formed which accumulated in the reaction mixture. The dissociation of prephenate allowed prephenate dehydrogenase to compete with prephenate dehydratase for the substrate.

Channel-Shuttle Mechanism for the Regulation of Phenylalanine and Tyrosine Synthesis at a Metabolic Branch Point in Pseudomonas aeruginosa

A bifunctional protein complex was partially purified from Pseudomonas aeruginosa. Catalytic activities for chorismate mutase and prephenate dehydratase coeluted from gel filtration and DEAE-cellulose chromatography columns. The protein complex had a molecular weight of approximately 134,000, as determined by gel filtration. In crude extracts or in partially purified preparations about one-half of the chorismate utilized by the complex is converted to phenylpyruvate, and the other half accumulates as prephenate. The chorismate mutase activity is strongly product-inhibited by prephenate, competitively with chorismate. Accordingly, the first reaction of the complex can be sufficiently retarded by prephenate so that all of the reaction product is phenylpyruvate. Chorismate mutase activity is also competitively inhibited by phenylalanine. Although phenylalanine is effective at low concentrations, maximal inhibition is only 50 to 60%. Inhibition of chorismate mutase by phenylalanine was completely lost after gel filtration. The prephenate dehydratase activity of the protein complex is nearly completely inhibited by 0.1 mm phenylalanine in either crude extracts or partially purified preparations. A second species of prephenate dehydratase was separated from the prephenate dehydratase-chorismate mutase aggregate by gel filtration or anion exchange chromatography. The second prephenate dehydratase had an estimated molecular weight of 76,000, a high affinity for prephenate, and was insensitive to feedback inhibition by phenylalanine. The physiological role of the latter enzyme is uncertain. The other regulatory enzymes of tyrosine and phenylalanine biosynthesis, prephenate mutase aggregate by gel filtration or anion exchange chromatography. The other regulatory enzymes of tyrosine and phenylalanine biosynthesis, prephenate dehydrogenase (molecular weight of 120,000) and 3-deoxy-d-arabino-heptulosonate-7-phosphate synthetase (molecular weight of 52,000), elute from Sephadex G-100 columns as fractions which are distinct from both the chorismate mutase-prephenate dehydratase complex and from the low-molecular-weight species of prephenate dehydratase. A shuttle mechanism governing the metabolic fate of prephenate (to phenylalanine or to tyrosine) is proposed in the context of a model which also accommodates several previously puzzling findings: (i) the dominating role of tyrosine in the control of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthetase and (ii) the lack of feedback control of prephenate dehydrogenase by tyrosine.

Chorismate Mutase-Prephenate Dehydratase from Escherichia coli K-12

Abstract The results of kinetic investigations with pure chorismate mutase-prephenate dehydratase from Escherichia coli K-12 were as follows: a pH optimum of 7.3 for the mutase activity, a Km of 0.045 mm for chorismate, and a Km of 1.0 mm for prephenate. l-Phenylalanine inhibited both the mutase and the dehydratase activities. In each case the relationship between activity and concentration of l-phenylalanine was sigmoidal and analysis of the results by the Hill equation gave values of n' = 2.3. d-Phenylalanine and l-tryptophan did not inhibit either activity whilst varying degrees of inhibition were observed with o-, m-, and p-monosubstituted fluoro-, chloro-, and hydroxyphenylalanines. Investigations with other phenylalanine analogues indicated that the α-NH2 group is not essential for inhibition, although it is required for maximal effect. There is an absolute requirement for an unmodified α-COOH group. Thiol compounds such as β-mercaptoethanol and dithioerythritol have little effect on the mutase activity but stimulate the dehydratase activity by approximately 150%.

Chorismate Mutase-Prephenate Dehydratase: PHENYLALANINE-INDUCED DIMERIZATION AND ITS RELATIONSHIP TO FEEDBACK INHIBITION

Abstract The bifunctional enzyme chorismate mutase-prephenate dehydratase catalyzes the first two reactions specific for phenylalanine biosynthesis in Salmonella typhimurium. Both activities are subject to feedback inhibition by phenylalanine. In the presence of phenylalanine the sedimentation coefficient of the enzyme is increased from approximately 5.6 to 8.0 S and the molecular weight is increased from approximately 109,000 to 220,000. This apparent dimerization is dependent upon the concentrations of phenylalanine and enzyme. Chorismate mutase-prephenate dehydratase that has been rendered insensitive to feedback inhibition either chemically or by mutational alteration does not dimerize. Therefore, dimerization appears somehow related to end product control. However, dimerization is not obligatory for feedback inhibition of either activity. At low protein concentration some enzyme does not dimerize in the presence of phenylalanine. Such enzyme can still be inhibited by phenylalanine. It is therefore suggested that phenylalanine binds to the monomer (smallest active species) to produce an inhibited monomer which dimerizes at appropriate protein concentrations.

Phenylalanine and tyrosine biosynthesis in Escherichia coli K-12: mutants derepressed for 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (phe), 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A.

Mutant strains of Escherichia coli have been isolated in which the synthesis of 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (phe) is derepressed, in addition to those enzymes of tyrosine biosynthesis previously shown to be controlled by the gene tyrR. The major enzyme of the terminal pathway of phenylalanine biosynthesis chorismate mutase-prephenate dehydratase is not derepressed in these strains. Genetic analysis of the mutants shows that the mutation or mutations causing derepression map close to previously reported tyrR mutations. A study of one of the mutations has shown it to be recessive to the wild-type allele in a diploid strain. It is proposed that the tyrR gene product is involved in the regulation of the synthesis of DAHP synthetase (phe) as well as the synthesis of DAHP synthetase (tyr), chorismate mutase-prephenate dehydrogenase, and transaminase A.